Mechanisms of ribosome stalling by SecM at multiple elongation steps
Translation regulation , as an essential component of the gene expression control , is usually mediated by a variety of ribosome-interacting factors and could take place at different stages during the translation cycle ( Duval et al . , 2015; Hinnebusch and Lorsch , 2012; Keiler , 2015; Spriggs et al . , 2010; Starosta et al . , 2014 ) . Furthermore , protein synthesis can also be modulated by regulatory nascent peptides , as demonstrated by an increasing number of such sequences found throughout all kingdoms of life ( Cruz-Vera et al . , 2011; Lovett and Rogers , 1996; Ramu et al . , 2009 ) . One such example is a variety of ribosome arrest peptides , often encoded by upstream open reading frames ( uORF ) or mRNA 5’ leaders that are utilized to regulate the expression of co-transcribed genes ( Cruz-Vera et al . , 2011; Ito and Chiba , 2013; Morris and Geballe , 2000; Wethmar , 2014 ) . Ribosome stalling by nascent peptides involves interactions of the regulatory peptide with the peptide exit channel on the 50S subunit , and in a few cases , requires coordination of small-molecule ligands , such as antibiotic erythromycin and tryptophan for the ErmCL and the TnaC-induced stalling , respectively ( Arenz et al . , 2014a; Bischoff et al . , 2014; Johansson et al . , 2014; Seidelt et al . , 2009 ) . In Escherichia coli , SecM-mediated translation stalling is specifically employed to regulate the secretion pathway of the cell ( McNicholas et al . , 1997; Nakatogawa and Ito , 2001; Oliver et al . , 1998 ) . SecM is encoded by the 5’-end half of a bicistronic mRNA , secM-secA . SecA is an ATPase-dependent molecular motor helping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria ( Bauer et al . , 2014; Economou and Wickner , 1994; Lill et al . , 1989; van der Wolk et al . , 1997 ) . SecM is a secreted protein with 170 amino acids in length , including a 17-amino-acid stalling sequence 150FSTPVWISQAQGIRAGP166 near its C terminus , which alone is sufficient to induce stalling . The intergenic region of the mRNA between secM and secA can form a stem-loop secondary structure , which would mask the translation initiation Shine-Dalgarno ( SD ) sequence of secA and therefore limits the synthesis of secA to a basal level . SecM-induced stalling could occur , as a consequence of impaired secretion pathways , and subsequently causes destabilization of the intergenic mRNA structure and increases the exposure of secA SD sequence to the ribosome ( Butkus et al . , 2003; Kiser and Schmidt , 1999; McNicholas et al . , 1997; Nakatogawa and Ito , 2001 , 2002; Sarker and Oliver , 2002 ) . Therefore , as a response to compromised secretion activity in the cell , the synthesis of SecA is upregulated . The stalling sequence of SecM could also induce ribosome stalling under normal conditions , but it is only temporary and quickly rescued by the functional Sec system through a simple pulling force by translocon ( Butkus et al . , 2003; Goldman et al . , 2015 ) . Thus , the regulatory peptide within SecM offers a feedback loop on the ribosome to ensure sufficient level of SecA in bacteria to regulate protein secretion . Previous biochemical and structural studies have demonstrated that the ribosome stalling originates from the interaction of the 17-amino-acid nascent peptide of SecM with the 50S exit tunnel components . In the arrest sequence , R163 , G165 , and P166 are essential , because mutation of any of these residues can completely abolish stalling ( Nakatogawa and Ito , 2002; Yap and Bernstein , 2009 ) . Other five residues ( F150 , W155 , I156 , G161 , and I162 ) are also important as mutations of them can abolish stalling partially ( Nakatogawa and Ito , 2002; Yap and Bernstein , 2009 ) . A few ribosomal components lining the tunnel are also required for efficient stalling , for mutations of A2058G , A2062U , or A2503G , or insertion of one adenine nucleotide within the five consecutive adenine residues ( A749-A753 ) , as well as mutations or deletion of selected residues from uL22 and uL4 could all alleviate translational stalling to certain extents ( Lawrence et al . , 2008; Nakatogawa and Ito , 2002; Vazquez-Laslop et al . , 2010; Woolhead et al . , 2006 ) . Previous structural studies of the SecM-arrested ribosome suggested that the interaction between the exit tunnel and the arrest peptide could change the conformation of the PTC ( peptidyl-transferase center ) to slow down the peptide bond formation ( Bhushan et al . , 2011; Gumbart et al . , 2012 ) . However , the previous structures were not in sufficient resolution for direct visualization of the atomic interactions between the tunnel components and the nascent peptide . Furthermore , a recent study employed fluorescence resonance energy transfer ( FRET ) to monitor the real-time translation of SecM on the ribosome ( Tsai et al . , 2014 ) , and revealed that the stalling is a dynamic process involving reduced elongation rates at a range of positions on the SecM mRNA , from G165 to 4–5 codons after the terminal P166 of the arrest sequence , including increased lifetime for both unrotated and rotated ribosomes at these codon positions . Nevertheless , although the stalling induced by SecM is not strictly a single-site event , G165 is the first predominant site of stalling ( Tsai et al . , 2014 ) . Recent advancement of cryo-EM single particle technique , such as the application of direct electron detection devices and efficient algorithms for conformational sorting of particles allow simultaneous high-resolution structural determination of several functional states from a single heterogeneous dataset ( Bai et al . , 2015; Cheng , 2015; Cheng et al . , 2015 ) . Therefore , we set out to use this method to analyze the structures of the ribosomes stalled on SecM mRNA . Our structural data of the two predominant forms of stalled ribosomes , one in post-state , and the other in hybrid rotated state , indicate that a collection of interactions between SecM and the exit tunnel cooperatively induce conformational changes of the PTC , leading to translation arrest at distinct elongation steps , including peptide-bond formation and tRNA translocation .
To understand the molecular mechanism of SecM-dependent translational stalling , we set out to purify SecM-stalled ribosome nascent chain complexes ( RNCs ) using an in vitro translation system from E . coli and to analyze their structures using single particle cryo-EM technique . To facilitate biochemical characterization and purification , two similar constructs were prepared: one encodes , from the N- to C-terminus , a 2xStrep-TEV-tag , the N-terminal 40 residues of OmpA , a Myc-tag , SecM stalling sequence ( residues 150–166 ) and tandem stop codons ( SEC-STOP ) ; the other contains an additional 6X-His-tag ( SEC-HIS-STOP ) after SecM stalling sequence> ( Figure 1— figure supplement 1A ) . After incubation of the plasmids with the S30-T7-based in vitro translation system , the presence and the amount of arrested peptidyl-tRNA can be detected using Western blot with primary antibody against Myc-tag ( Figure 1— figure supplement 1B–E ) . We found that the peptidyl-tRNA in the reaction mixture started to accumulate after 5 min , peaked at 15 min and became diminished after 30 min . Therefore , we chose 15 min as the incubation time to purify SecM-stalled RNCs . The design of the SEC-HIS-STOP construct was to assess how frequent the ribosome would go beyond P166 . As shown in Figure 1— figure supplement 1B , lower band corresponding to SecM-HIS peptide also existed in the reaction mixtures , in a comparable amount to the peptidyl-tRNA , indicating the ribosome was able to go beyond SecM coding sequence and reach the stop codon with increasing incubation time . However , after treatment of the samples with excessive RNase A , which destroys all RNAs and releases the peptide from the peptidyl-tRNA , only the band of SecM peptide , but not SecM-HIS , sharply increased , indicating that the peptidyl-tRNA in the reaction mixtures is indeed arrested on the very 3-end of SecM coding sequence . The construct of SEC-STOP was specifically used to further synchronize the RNCs on the terminal codon to provide a homogeneous complex for structural analysis . As designed , the peptidyl-tRNA in the reaction mixture of SEC-STOP displayed exactly the same motility on the gel as the one from SEC-HIS-STOP . The RNCs were isolated by a sucrose cushion followed by Strep-affinity chromatography , and further separated and enriched by a second round of sucrose cushion ( Figure 1— figure supplement 1C , D ) . Similar as previous studies of ribosome stalling ( Bhushan et al . , 2011; Frauenfeld et al . , 2011; Halic et al . , 2006; Yap and Bernstein , 2009 ) , chloramphenicol was added in the sucrose cushion buffer to stabilize the RNC and to minimize the peptidyl-tRNA hydrolysis during the lengthy purification . As expected , Western blotting indicates that a clean single band of the peptidyl-tRNA was detected in the purified RNCs ( Figure 1— figure supplement 1D ) , indicating the compositional homogeneity of the purified RNC . To identify nascent peptides in the purified RNCs , we further purified nascent peptides from SecM-stalled RNCs ( Figure 1— figure supplement 2A , B ) and subjected them to mass spectrometry analysis . The C-terminal sequencing of nascent peptides using a tandem MS/MS ( Figure 1— figure supplement 2C–F ) revealed two species , with their C-termini ending at G165 and P166 , respectively . Next , we applied cryo-EM to analyze purified SecM-RNCs . A cryo-EM data set , composed of 238 , 212 particles , were subjected to maximum-likelihood-based 2D and 3D sorting ( Scheres , 2010 , 2012 ) , and as a result ( Figure 1— figure supplement 3 ) , two cryo-EM density maps , corresponding to the two major populations of the data , one with the ribosome in classical unrotated state ( a peptidyl-tRNA at the P-site ) , the other with the ribosome in rotated state ( two hybrid tRNAs ) , were obtained at resolution of 3 . 7 and 3 . 3 Å , respectively ( Figure 1A , B and Figure 1— figure supplement 4A–D ) . These two maps have well-resolved densities for the ribosome and tRNA , with a clear separation of most of the nucleotide bases and protein sidechains ( Figure 1— figure supplement 4E–H ) . To further improve the density quality of the nascent chains , a soft mask of the 50S subunit ( plus tRNA ) was applied during refinement ( Supplementary file 1 ) , similarly as previously described ( Amunts et al . , 2014; Brown et al . , 2014; Greber et al . , 2014a; Greber et al . , 2014b; Voorhees et al . , 2014 ) , which resulted in slightly improved overall resolutions ( Figure 1— figure supplement 4A , D ) , but considerably enhanced densities for the nascent chains . The improved structures allowed us to trace and de novo model the nascent chains within the tunnel ( Video 1–3 ) ( see ‘Materials and methods for model validation details ) . 10 . 7554/eLife . 09684 . 003Figure 1 . Cryo-EM structures and atomic models of SecM-stalled ribosomes . ( A ) Cryo-EM density map ( 3 . 6 Å ) and atomic model of SecM-Gly-RNC , with a peptidyl-tRNA at the P/P-site . Surface representation of the map ( 50S , 30S , SecM-tRNA , nascent chain , and mRNA in light blue , yellow , red , green , and orange , respectively ) is shown in the middle panel , and the atomic model on the right panel . The cut-away view of the density map on the left panel highlights the tunnel and the nascent peptide within . ( B ) Same as ( A ) , but for SecM-Pro-RNC ( 3 . 3 Å ) . The A/P*-site tRNA ( SecM-tRNA ) and P/E-site tRNA are colored forest green and bright yellow , respectively . For atomic models , mRNA , 16S rRNA , 30S proteins , 23S rRNA , and 50S proteins are colored orange , cyan , blue , grey , and magenta , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 00310 . 7554/eLife . 09684 . 004Figure 1—figure supplement 1 . Preparation of SecM-stalled RNCs . ( A ) Schematic diagrams showing the two experimental constructs , SecM-HIS-STOP and SEC-STOP , used for in vitro translation . ( B ) Western blot ( using anti-Myc antibody ) analysis of the in vitro translation reactions . The reactions were performed with either of the two constructs for indicated time and treated with ( + ) or without ( - ) RNase A after reaction . The reaction solutions were resolved on NuPAGE and examined by Western blot analysis . The positions of the peptidyl-tRNA ( peptidyl-tRNA ) , rescued SecM polypeptide ( peptide-his ) , and hydrolyzed SecM peptide ( peptide ) by RNase A are indicated . ( C ) NuPAGE examination of RNC purification . SecM-stalled RNCs with the construct of SEC-STOP were isolated by a sucrose cushion ( first supernatant and first pellet ) , followed by a Strep-affinity chromatography ( flowthrough and elution ) and further purified by a second round of sucrose cushion ( second supernatant and second pellet ) . Fractions of RNCs obtained from the second pellet were also treated with RNase A . The asterisk indicates the band of added RNase A . ( D ) Western blot ( using anti-Myc antibody ) analysis of the NuPAGE as shown in C . As shown , after the second pelleting , only one major band for the peptidyl-tRNA is present , which , upon addition of RNase A becomes free SecM peptide . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 00410 . 7554/eLife . 09684 . 005Figure 1—figure supplement 2 . Mass spectrometry analysis of the nascent peptides purified from SecM-stalled RNCs . ( A ) NuPAGE examination of nascent peptide purification . SecM-stalled RNCs were treated with 10 mM EDTA and 100 U of RNAse A to disassemble ribosomes and to digest ribosomal RNA ( RNC + RNAse A ) , and then enriched by Strep-affinity chromatography ( flowthrough and washing ) . Eluted peptide was loaded at different volumes to test the purity . ( B ) Western blot ( using anti-Myc and anti-Strep antibody ) analysis of the purified nascent peptides from NuPAGE as shown in A . ( C-F ) Respective first-order ( C-D ) and second-order ( E-F ) mass spectra of digested nascent peptides . Two species of peptides ( digested by lysyl endopeptidase ) starting from F150 could be identified from their first-order mass spectra ( C-D ) . The ratio of proline to glycine ( C-terminal ) is about 2:1 . ( E-F ) The MS/MS spectra of respective two peptide species . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 00510 . 7554/eLife . 09684 . 006Figure 1—figure supplement 3 . Overview of the image processing . ( A ) A representative raw micrograph of SecM-stalled ribosomes . A Fourier transform of the micrograph is also shown . ( B ) Representative 2D class averages from reference-free classification . ( C ) . Workflow of the 3D classification ( see ‘Methods’ for details ) . To improve the density quality of the nascent polypeptides , a soft mask of the 50S subunit and tRNAs ( purple circle ) was applied during further structural refinement . The A-site tRNA , P-site tRNA , A/P-site tRNA , P/E-site tRNA , and nascent polypeptide are colored in blue , red , forest green , yellow , and green , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 00610 . 7554/eLife . 09684 . 007Figure 1—figure supplement 4 . FSC curves and Cryo-EM density . ( A , C ) Gold-standard Fourier shell correlation curves of the reconstructions of SecM-Gly-RNC ( A ) and SecM-Pro-RNC ( C ) with a soft 70S mask ( black ) or a 50S mask ( red ) applied during refinement . ( B , D ) The local resolution maps of the 50S-masked SecM-Gly-RNC ( B ) and SecM-Pro-RNC ( D ) . Throughout much of the interior , the local resolution is significantly better than the overall resolution . ( E-H ) Examples of the densities in the cryo-EM map of SecM-Gly-RNC , including anα helix ( residues 53–71 from protein bL20 , E ) , a β strand ( residues 33–38 from protein uL30 , F ) , nucleotide bases ( 576–581 from 23S rRNA , G ) , and the P-site tRNA ( H ) . The models were colored as forest green by elements , and the density maps were showed as dark mesh . The densities of nucleotide bases and amino acids are clearly separated from each other , in good agreement with reported resolutions . The density map was displayed at ∼5σ contour level . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 00710 . 7554/eLife . 09684 . 008Figure 1—figure supplement 5 . Map density of the antiodon region of tRNAs and codon region of mRNA in SecM-stalled RNCs . ( A ) Local density of the P-site codon of mRNA in SecM-Gly-RNC . ( B ) Local density of the P-site codon of mRNA in SecM-Pro-RNC . ( C ) Same as B , but for the A-site codon . ( D-F ) Same as A–C , but for the anticodon regions of respective tRNAs in the two map . The density map is displayed at ∼5σ contour level . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 00810 . 7554/eLife . 09684 . 009Figure 1—figure supplement 6 . Positions of tRNAs in SecM-stalled RNCs . ( A-B ) Comparison of the tRNA in SecM-Gly-RNC ( red ) with the tRNAs in classical A , P , and E-state ( PDB 4V6F , gray ) ( Jenner et al . , 2010 ) . The alignment was done using the 50S subunit ( the 23S rRNA residues 1600–2700 ) ( A ) or 30S subunit body ( the 16S rRNA residues 10–550 ) ( B ) as reference . ( C-D ) Comparison of the tRNAs in SecM-Pro-RNC ( yellow , forest green ) with the tRNAs in classical states ( gray ) . The alignment was done using the 50S subunit ( the 23S rRNA residues 1600–2700 ) ( C ) , the 30S subunit body ( the 16S rRNA residues 10–550 ) ( D , upper panel ) or the 30S subunit head ( the 16S rRNA residues 920–1400 ) ( D , lower panel ) as reference . ( E ) The P/E-tRNA in SecM-Pro-RNC ( yellow ) , the P/E-tRNA ( PDB ID 4V9D , blue ) ( Dunkle et al . , 2011 ) , and the pe/E-tRNA ( PDB ID 4W29 , purple ) ( Zhou et al . , 2014 ) were aligned using the 30S body ( the 16S rRNA residues 10–550 ) as reference . ( F-G ) The A/P*-tRNA in SecM-Pro-RNC ( forest green ) , the A/P-tRNA ( PDB ID 4V7C , orchild ) , and the A/P*-tRNA ( PDB ID 4V7D , cyan ) ( Brilot et al . , 2013 ) were aligned using the 30S head ( the 16S rRNA residues 920–1400 ) as reference . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 00910 . 7554/eLife . 09684 . 010Figure 1—figure supplement 7 . Chloramphenicol is present in SecM-Pro-RNC , but not SecM-Gly-RNC . ( A ) The map density for selected region of the PTC in SecM-Gly-RNC is shown in mesh representation , superimposed with the atomic model . As shown , superimposition of chloramphenicol ( CHL ) ( PDB ID 4V7T ) ( Dunkle , et al . , 2010 ) indicates that there is no chloramphenicol bound in SecM-Gly-RNC . ( B ) Same as ( A ) , but for SecM-Pro-RNC . At expected chloramphenicol-binding position , clear densities of chloramphenicol could be located . The density map is displayed at ∼5σ contour level . The alignment was done using the 50S subunit ( the 23S rRNA residues 2400–2800 ) as reference . The RMS deviations between respective reference sequences are 1 . 5 Å for SecM-Gly-RNC vs 4V7T , and 1 . 1 Å for SecM-Pro-RNC vs 4V7T . ( C ) A snapshot of the binding environment of chloramphenicol ( orange ) in the crystal structure of chloramphenicol-bound 70S ribosome ( PDB ID 4V7T ) ( Dunkle et al . , 2010 ) . Potential hydrogen bonds are shown in black dashed lines . ( D ) Same as C , but for SecM-Pro-RNC . Note that chloramphenicol interacts with different nucleotides , contrasting the structural observations from the crystal structure ( PDB ID 4V7T ) ( Dunkle et al . , 2010 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 01010 . 7554/eLife . 09684 . 011Figure 1—figure supplement 8 . Cross-validation of the atomic model refinement . Cross-validation of model refinements for SecM-Gly-RNC ( A ) and SecM-Pro-RNC ( B ) . Refer to methods for details . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 01110 . 7554/eLife . 09684 . 012Video 1 . Cryo-EM density map of SecM-Gly-RNC . The cryo-EM density map of SecM-Gly-RNC is shown in transparent surface representation . The 50S subunit , 30S subunit , SecM-Gly-tRNA , and mRNA are colored pale blue , yellow , red , and orange , respectively . In the final movie frames , the map is shown in a cut-away view and zoomed into the region of the peptide exit tunnel , highlighting the densities for the nascent chain . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 01210 . 7554/eLife . 09684 . 013Video 2 . Cryo-EM density map of SecM-Pro-RNC . The cryo-EM density map of SecM-Pro-RNC is shown in transparent surface representation . The 50S subunit , 30S subunit , SecM-Pro-tRNA ( A/P* ) , P/E-tRNA , and mRNA are colored pale blue , yellow , green , bright yellow , and orange , respectively . In the final movie frames , the map is shown in a cut-away view and zoomed into the region of the peptide exit tunnel , highlighting the densities for the nascent chain . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 01310 . 7554/eLife . 09684 . 014Video 3 . Atomic models of the nascent peptides in SecM-Gly-RNC and SecM-Pro-RNC . Segmented density maps of the peptidyl-tRNAs in SecM-Gly-RNC ( red ) and in SecM-Pro-RNC ( green ) are shown in surface representation , with C-terminal half of SecM peptide highlighted in zoom-in views . Atomic models of the nascent peptides are superimposed with the density maps , with a few landmark residues of SecM labeled . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 014 Based on the information from mass spectrometry and structural modeling of the two density maps , the two structures were termed as SecM-Gly-RNC and SecM-Pro-RNC according to the identity of the peptidyl-tRNA ( Gly or Pro ) in the RNCs ( Figure 1—figure supplement 3 ) , following the same notion in the previous work ( Bhushan et al . , 2011 ) . This assignment of the two maps was supported by two sets of structural observations . First , the local densities of the mRNA: tRNA duplex at the PTC in the two maps could be best explained by our assignment , because the anticodon of tRNApro ( GGA ) is composed of purines exclusively , in contrast to that of tRNAgly ( CCG ) . In our map of SecM-Pro-RNC , bases of the three A-site anti-codon residues indeed appears to be relatively larger ( Figure 1—figure supplement 5F ) , compared with those in SecM-Gly-RNC ( Figure 1—figure supplement 5D ) . Vice versa , analysis of local density in the A- and P-site codons of the mRNAs in the two maps also supports our assignment ( Figure 1—figure supplement 5A–C ) . Second , two bulky residues ( W155 and F150 ) within SecM arrest peptide could be readily located in the exit channel of the two density maps , and their positions are incompatible with other possible assignments . The peptidyl-tRNA in SecM-Gly-RNC is at the classical P site ( P/P state ) ( Figure 1—figure supplement 6A , B ) , while the two tRNA in SecM-Pro-RNC are at hybrid states ( Figure 1—figure supplement 6C–G ) . In SecM-Pro-RNC , the peptidyl-tRNA is at a position similar to previously reported A/P* hybrid state ( Brilot et al . , 2013 ) ( Figure 1—figure supplement 6F , G ) , and the other tRNA is at a position between P/E and pe/E hybrid states ( Polikanov et al . , 2014 ) ( Figure 1—figure supplement 6E ) , closer to the P/E configuration . In the dataset , the hybrid state of SecM-Pro-RNC accounts for 36% of total particles , with a ratio of 1:1 . 5 for SecM-Pro-RNC ( rotated ) : SecM-Gly-RNC ( unrotated ) . Also comparable to the previous observation ( Bhushan et al . , 2011 ) , a minor class of particles resulted in a low-resolution structure of a pre-state ribosome ( unrotated with A/A and P/P-tRNAs ) , likely representing SecM-Gly-RNC with prolyl-tRNA at the A-site ( Figure 1—figure supplement 3 ) . It should be noted that during a normal cycle of elongation , the fraction of rotated ribosomes is much less ( Fischer et al . , 2010; Kim et al . , 2007; Munro et al . , 2007 ) , as it reflects a high-energy state and requires stabilization from translation factor ( Frank et al . , 2007 ) . Therefore , the increased lifetime of the A/P* , P/E intermediate states of tRNAs in SecM-Pro-RNC suggests this structure might represent a stalled form that is trapped in an intermediate translocational state . In SecM-Gly-RNC , extensive interactions between the nascent chain and the tunnel could be readily identified ( Figure 2A ) . The first region involves the PTC , the CCA end of the peptidyl-tRNA and R163 of SecM . The CCA-end of the P-tRNA forms canonical base pairing with the P-loop ( Figure 2B ) ; the base of U2585 contacts the peptidyl bond linkage between G165 and A76 of the P-site tRNA ( Figure 2C ) ; R163 is situated at a space confined by three nucleotides , U2506 , A2451 , and C2452 ( Figure 2D ) . These extensive interactions involving R163-G165 perfectly explain their critical roles in SecM-induced stalling ( Nakatogawa and Ito , 2002; Yap and Bernstein , 2009 ) . Immediately below R163 along the tunnel , strong density connections can be observed between the carbonyl oxygen of I162 and the sugar ring of G2505 , and between the sidechain of I162 and the base of C2610 ( Figure 2E ) . Further downstream , multiple interactions between Q160-Q158 and U2609 , C2610 , C2611 , and A2062 could be identified ( Figure 2E and Figure 2—figure supplement 1A , B ) , consistent with previous mutational data that perturbation of this interface either at the tunnel ( A2062U ) or the nascent peptide ( Q160P ) impairs translation stalling ( Vazquez-Laslop et al . , 2010; Woolhead et al . , 2006; Yap and Bernstein , 2009 ) . In the mid-tunnel region , the sidechain of W155 interacts with the side-chain of R61 of uL4 ( Figure 2F ) , and the backbone of I156-W155-S154 is also close to several tunnel components , including A751 and G91-R92 of uL22 ( Figure 2F and Figure 2—figure supplement 1C ) . These observations could well explain the previous finding that mutations of either W155 or I156 into alanine can rescue the SecM-induced stalling ( Nakatogawa and Ito , 2002 ) . In the lower region of the tunnel , a strong hydrophobic contact between F150 and A93 of uL22 is evident ( Figure 2G ) . Again , the contribution of uL22 residues ( G91 and A93 ) in this region to the stalling was previously demonstrated by mutational data ( Lawrence et al . , 2008 ) . Lastly , beyond F150 , the last engineered residue of SecM , two sets of contacts between the Myc-tag and the tunnel could also be observed . One involves K84-I85 of uL22 and E7-I5 of the Myc sequence ( Figure 2G ) , and the other one involves E1-K3 of the Myc sequence , A508 and A1321 of the 23S rRNA ( Figure 2—figure supplement 1D ) . The N-terminal sequences of SecM beyond F150 are poorly conserved and not essential for stalling . However , previous data showed that SecM150-166 is less efficient in stalling than SecM140-166 ( Nakatogawa and Ito , 2002 ) , indicating that the interactions beyond F150 as we observed here , although might not be sequence-specific , also contributes to the stalling . 10 . 7554/eLife . 09684 . 015Figure 2 . Interactions between SecM-Gly-nascent chain and ribosomal tunnel . ( A ) A zoom-in view of the density map in the regions where SecM-Gly-nascent chain interacts with exit tunnel . A transverse-section of the cryo-EM map of the SecM-Gly-RNC , showing P-tRNA and SecM-Gly-nascent chain within the ribosomal exit tunnel is provided in the lower right corner . ( B–G ) Zoom-in views of the density map in selected regions , highlighting extensive interactions between SecM and ribosomal tunnel components . Map density is shown in mesh , and the atomic model in stick representation . The coloring scheme is the same as in ( A ) . Strong interactions between SecM-nascent-peptide and ribosomal components are indicated by red asterisks . The primary sequences from N-terminal Myc-tag to C-terminal of SecM peptide within the exit tunnel are shown below the panel ( F ) . To illustrate the interaction between the nascent peptide and ribosomal tunnel components , the density map was displayed at relatively lower contour ( 1 . 5–3 σ ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 01510 . 7554/eLife . 09684 . 016Figure 2—figure supplement 1 . Selected regions of interactions between SecM and ribosomal tunnel components in SecM-Gly-RNC . ( A–D ) Zoom-in views of the density map in selected regions , highlighting extensive interactions between SecM and ribosomal tunnel components . The coloring scheme is the same as in Figure 2 . The interactions between SecM-nascent-peptide and ribosomal components are indicated by red asterisks . The primary sequences from N-terminal Myc-tag to C-terminus of the SecM peptide are shown below the panel ( D ) . To illustrate the interaction between the nascent peptide and ribosomal tunnel components , the density map was displayed at relatively lower contour levels ( 1 . 5–3σ ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 016 Most of the residues of SecM contributing to interact with the tunnel in SecM-Gly-RNC also extensively interact with the tunnel in SecM-Pro-RNC , but with completely different patterns ( Figure 3A ) . At the PTC , the CCA-end of the A/P*-tRNA is not orientated for canonical base pairing between C74-C75 of the peptidyl-tRNA and G2252-G2251 of the P-loop ( Figure 3B ) . A strong density connection is seen between the base of U2585 and P166 ( Figure 3C ) , which might be responsible for the distorted conformation of the CCA-end of the A/P*-tRNA . Compared with SecM-Gly-RNC , with addition of one more amino acid , G165 moves into a pocket formed by the bases of U2506 and A2451 ( Figure 3—figure supplement 1A ) , while R163 relocates its sidechain between the bases of U2586 and U2609 ( Figure 3D ) . In addition , strong density connections can be observed between the sidechain of I162 and the base of A2062 ( Figure 3D , E ) . Further downstream , Q160 and Q158 , through their sidechains , form hydrogen bonds with the bases of U2609 and A752 , respectively ( Figure 3—figure supplement 1B , C ) . Within the mid-tunnel region , the sidechain of R61 of uL4 , instead of interacting with W155 in SecM-Gly-RNC , is at a distance capable of hydrogen bonding with the carbonyl oxygen of I156 ( Figure 2F and Figure 3—figure supplement 1D ) . The sidechain of W155 , in contrast to its location in SecM-Gly-RNC , flips ∼90° and stacks with the base of A751 ( Figure 3F and Figure 3—figure supplement 1D ) . The pattern of interactions at F150 and beyond is very similar with slight variation ( Figure 3G and Figure 3—figure supplement 1E ) . F150 still maintains hydrophobic interaction with A93 of uL22 ( Figure 3G ) , but the distance is significantly larger than that in SecM-Gly-RNC . The C- and N-terminal sequences of the Myc-tag interact similarly with K84-I85 of uL22 and A1321 of the 23S rRNA , respectively ( Figure 3G and Figure 3—figure supplement 1E ) . 10 . 7554/eLife . 09684 . 017Figure 3 . Interactions between SecM-Pro-nascent chain with ribosomal tunnel . ( A ) A zoom-in view of the density map in the region where SecM-Pro-nascent chain interacts with ribosomal tunnel . A transverse-section of the cryo-EM map of the SecM-Pro-RNC , showing P-tRNA and SecM-Pro-nascent chain within the ribosomal exit tunnel is provided in the lower right panel . ( B–G ) Zoom-in views of the density map in selected regions , highlighting extensive interactions between SecM and ribosomal tunnel components . Map density is shown in mesh , and the atomic model in stick representation . The coloring scheme is the same as in ( A ) . Strong interactions between SecM-nascent-peptide and ribosomal components are indicated by red asterisks . The primary sequences from N-terminal Myc-tag to C-terminal of SecM peptide within the exit tunnel are shown below the panel ( F ) . To illustrate the interaction between the nascent peptide and ribosomal tunnel components , the density map was displayed at relatively lower contour ( 1 . 5–3 σ ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 01710 . 7554/eLife . 09684 . 018Figure 3—figure supplement 1 . Selected regions of interactions between SecM and ribosomal tunnel components in SecM-Pro-RNC . ( A–E ) Zoom-in views of the density map in selected regions , highlighting extensive interactions between SecM and ribosomal tunnel components . Map density is shown in mesh , and the atomic model in stick representation . The coloring scheme is the same as in Figure 3 . The interactions between SecM-nascent-peptide and ribosomal components are indicated by red asterisks . The primary sequences from N-terminal Myc-tag to C-terminus of the SecM peptide are shown below the panel ( D ) . To illustrate the interaction between the nascent peptide and ribosomal tunnel components , the density map was displayed at relatively lower contour levels ( 1 . 5–3σ ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 018 The atomic details in SecM-Pro-RNC are very different from those in SecM-Gly-RNC , especially for the C-terminal half of the arrest sequence . Actually , many of contacts observed in SecM-Pro-RNC have been previously predicted by a molecular dynamics simulation study ( Gumbart et al . , 2012 ) , indicating that SecM-Pro-RNC is likely a thermodynamically favored state . Through analysis of the two structures , there appears to be two common constrict sites on the tunnel wall , and the interactions at these locations in the two structures are highly residue-specific: one is formed by A751 of 23S rRNA and R61 of uL4 , and the other by A93 and K84 of uL22 . The presence of these specific constrict interactions in the two structures suggests that the bulky residues of SecM , W155 , and F150 in particular , function to contain the peptide to slow down its passage within the tunnel to posit N-terminal R163 , G165 , and P166 at specific locations to induce stalling . Peptide bond formation in the ribosome requires precise positioning of the peptidyl-tRNA and the A-tRNA ( Polikanov et al . , 2014; Schmeing et al . , 2005a; Schmeing et al . , 2005b ) . Highly conserved rRNA residues in the PTC adopt specific conformations in different functional states to determine the catalytic kinetics of the peptide bond formation . To understand how the interactions between SecM and ribosomal components observed in our models contribute to the translation stalling , we analyzed conformational differences of the PTC in SecM-Gly-RNC and SecM-Pro-RNC from the previously characterized induced ( PDB ID 1VQN ) and uninduced states of PTC ( PDB ID 1VQ6 ) ( Figure 4A , C ) ( Schmeing et al . , 2005b ) . These two states , obtained by structural determination of the active 50S subunit ( Schmeing et al . , 2005a; Schmeing et al . , 2005b ) or the ribosome ( Polikanov et al . , 2014 ) with different A-site and P-site tRNAs or analogues , reflect two major states of the PTC , inactive and active for peptide bond formation . When the PTC transits from the uninduced state to induced state , U2585 and U2584 would shift away by 3–4 Å to allow the A-tRNA accommodation ( Figure 4A ) , and U2506 would rotate toward the P-tRNA ( Schmeing et al . , 2005b; Voorhees et al . , 2009 ) ( Figure 4C ) . By comparing the conformation of PTC in SecM-Gly-RNC with those of the uninduced and induced states , we found that both U2585 ( Figure 4B ) and U2506 ( Figure 4D ) in SecM-Gly-RNC adopt positions similar as in the uninduced state ( Video 4 ) . Therefore , these interactions originated from G165-R163 are likely responsible for constricting the PTC of SecM-Gly-RNC , especially U2585 and U2506 , into an uninduced state that disfavors A-tRNA accommodation and subsequent peptide bond formation . Moreover , the extent of U2585 shifting is even more toward the inactive form of the uninduced state , with a 70° flip compared with that of the induced state ( Figure 4—figure supplement 1A ) . Similar observation is also true for U2506 in SecM-Gly-RNC ( Figure 4—figure supplement 1B ) . 10 . 7554/eLife . 09684 . 019Figure 4 . SecM stabilizes the PTC of SecM-Gly-RNC in an inactive state . ( A ) Conformational difference between the uninduced ( PDB ID 1VQ6 , plum ) ( Schmeing et al . , 2005a ) and the induced states ( PDB ID 1VQN , gray ) ( Schmeing et al . , 2005a ) of the PTC . Direction of the shift for U2585 between two states and corresponding distances are labeled . As shown in ( A ) , U2585 and U2584 would shift away by 3–4 Å from the uninduced state to the induced state . ( B ) U2584 and U2585 in SecM-Gly-RNC ( 23S rRNA in blue and peptidyl-tRNA in red ) shift away from their regular positions in the induced state ( grey ) and assume an uninduced state . ( C ) Rotation of U2506 accompanying the shift of U2585 as in ( A ) , upon transition from the uninduced ( plum ) to induced ( grey ) states . ( D ) Position and interaction of R163 in SecM-Gly-RNC . R163 leads to a rotation of U2506 to assume an uninduced conformation . ( E ) Comparison of A2602 in SecM-Gly-RNC with that of the uninduced and induced states . ( F ) The conformation of A2602 ( blue ) in SecM-Gly-RNC is incompatible with the binding of release factor2 ( RF2 ) to the PTC . The position of R163 ( red ) in SecM-Gly-RNC is in clash with the GGQ motif of RF2 ( bright yellow ) . The coordinates of RF2 is from a crystal structure of the 70S·RF2 complex ( PDB , ID 4V5J ) ( Jin et al . , 2010 ) . The alignment was done using the 23S rRNA residues 2400–2800 as reference . The RMS deviations between respective reference sequences are 0 . 3 Å for 1VQN vs 1VQ6 , 1 . 5 Å for SecM-Gly-RNC vs 1VQN , 1 . 0 Å for SecM-Gly-RNC vs 4V5J . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 01910 . 7554/eLife . 09684 . 020Figure 4—figure supplement 1 . Conformational changes of the PTC nucleotides in SecM-stalled RNCs . ( A ) Comparison of U2585 in SecM-Gly-RNC ( azure ) and SecM-Pro-RNC ( blue ) with the ribosomal structures in the induced ( PDB 1VQN , gray ) , uninduced ( PDB 1VQ6 , plum ) ( Schmeing et al . , 2005a; Schmeing et al . , 2005b ) , and chloramphenicol bound ( PDB 4V7T , orange ) ( Dunkle , et al . , 2010 ) states . U2585 in both SecM-Gly-RNC and SecM-Pro-RNC shifts away from their regular positions in the induced state to different extents . ( B ) Same as ( A ) , but for U2506 . U2506 in SecM-Gly-RNC adopts a position similar as in the uninduced state , and in SecM-Pro-RNC the induced state . ( C ) Same as A , but for A2602 . A2602in SecM-Gly-RNC is in the uninduced state , and in SecM-Pro-RNC flips away from the induced state . The alignment was done using the 23S rRNA residues 2400–2800 as reference . The RMS deviations between respective reference sequences are 0 . 3 Å for 1VQ6 vs 1VQN , 1 . 5 Å for SecM-Gly-RNC vs 1VQN , 1 . 4 Å for SecM-Pro-RNC vs 1VQN , and 1 . 1 Å for 4V7T vs 1VQN . As shown , comparison of our models with the chloramphenicol-bound one indicates that the conformations of the important bases ( U2506/U2585/A2602 ) of the PTC in our models are different from the crystal structure of ribosome bound with chloramphenicol , indicating that the stalling in our structures is not determined by chloramphenicol . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 02010 . 7554/eLife . 09684 . 021Video 4 . Comparison of the PTC in SecM-Gly-RNC with that of the induced state . Superimposition of the atomic model of SecM-Gly-RNC with that of the induced state ( 1VQN ) ( Schmeing et al . , 2005b ) in the PTC region . Morphing between two models was shown . Critical residues of SecM and the 23S rRNA are labeled . Also see Figure 7 and text for details . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 021 Similar to TnaC- and MifM-stalled ribosomes ( Bischoff et al . , 2014; Sohmen et al . , 2015 ) , A2602 in SecM-Gly-RNC adopts a position ( Figure 4E and Figure 4—figure supplement 1C ) , which would block the entry of the incoming aminoacyl-tRNA to the A-site of the PTC . This specific configuration of the PTC in SecM-Gly-RNC is also incompatible with the binding of release factors , as the GGQ motif of RF2 ( PDB ID 4V5J ) ( Korostelev et al . , 2008 ) sterically clashes with A2062 , as well as with R163 ( Figure 4F ) . These observations provide a possible explanation why SecM-stalled ribosomes are stable over an hour ( Evans et al . , 2005; Tsai et al . , 2014; Uemura et al . , 2008 ) , and resistant to the hydrolysis of peptidyl-tRNA by puromycin ( Muto et al . , 2006; Tsai et al . , 2014 ) . The geometry of the PTC in SecM-Pro-RNC also changed , but differently from SecM-Gly-RNC . First , U2585 shifts slightly toward the direction of the induced state , but still in a state closer to the uninduced conformation ( Figure 4—figure supplement 1A and Figure 5—figure supplement 1B ) . Local conformational difference at U2585 and U2586 is evidently due to the interactions with P166 and R163 of SecM ( Figure 5A , C ) . Second , In contrast to SecM-Gly-RNC , U2506 now assumes an induced state ( Figure 4—figure supplement 1B , Figure 5—figure supplement 1D ) . Third , the interaction between SecM-I162 and A2062 stabilizes the latter in a state different from the induced state ( Figure 5A , D ) . The most intriguing observation is that the base pairing between the CCA-end of A/P*-tRNA and the P-loop is disrupted ( Figures 3A , 5E , Video 5 ) . The base of C75 has a twist and is seen to extend toward the base of G2252 instead of G2251 ( Figure 5E ) , and C74 also deviates from its optimal base-pairing position with G2252 . The destabilization of the interaction between the A/P*-tRNA with the P-loop appears to result from a cascade of interactions described in the above . Specifically , R163 and P166 of SecM are likely responsible for preventing the establishment of a stable P-loop interaction in SecM-Pro-RNC . 10 . 7554/eLife . 09684 . 022Figure 5 . SecM destabilizes the interaction between CCA-end of the A/P*-tRNA and the P-loop in SecM-Pro-RNC . ( A ) Interactions between SecM nascent peptide and ribosomal components at the PTC in SecM-Pro-RNC . Peptidyl-tRNA and 23S rRNA are colored forest green and blue , respectively . ( B ) Comparison of the 23S rRNA nucleotides in SecM-Pro-RNC with that of the induced state of the PTC ( PDB 1VQN , cyan ) ( Schmeing et al . , 2005a ) . ( C–E ) Zoom-in views of ( B ) , with orientations optimized to show the conformational difference for U2585-U2586 ( C ) , A2062 ( D ) , and the P-loop ( E ) . The alignment was done using the 23S rRNA residues 2400–2800 as reference . The RMS deviation between respective reference sequences is 1 . 4 Å for SecM-Pro-RNC vs 1VQN . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 02210 . 7554/eLife . 09684 . 023Figure 5—figure supplement 1 . Conformation states of the PTC nucleotides in SecM-Pro-RNC around the PTC . ( A ) Conformational difference between the uninduced ( PDB ID 1VQ6 , plum ) ( Schmeing et al . , 2005b ) and the induced states ( PDB ID 1VQN , gray ) ( Schmeing et al . , 2005b ) of the PTC . Direction of shift for U2585 and corresponding distances are labeled . As shown in ( A ) , U2585 and U2584 would shift away by 3–4 Å from the uninduced state to the induced state . ( B ) U2585 in SecM-Pro-RNC ( blue ) shifts slightly toward the direction of the induced state ( gray ) , but still in a state closer to the uninduced conformation ( plum ) . ( C ) Rotation of U2506 accompanying the shift of U2585 as in ( A ) , upon transition from the uninduced ( plum ) to induced ( grey ) states . ( D ) Position and interaction of G165 in SecM-Pro-RNC . The alignment was done using the 23S rRNA residues 2400–2800 as reference . The RMS deviations between respective reference sequences are 0 . 3 Å for 1VQN vs 1VQ6 ( A , C ) , and 1 . 4 Å for SecM-Pro-RNC vs 1VQN ( B , D ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 023 Taken together with the kinetic data that SecM increases the lifetime of rotated state of the ribosome ( Tsai et al . , 2014 ) , our structural data on SecM-Pro-RNC therefore suggest that SecM might also impair the hybrid peptidyl-tRNA accommodation to the P-loop to slow down the tRNA translocation , in addition to its inhibition on peptide bond formation in SecM-Gly-RNC . 10 . 7554/eLife . 09684 . 024Video 5 . Comparison of the PTC in SecM-Pro-RNC with that of the induced state . Superimposition of the atomic model of SecM-Pro-RNC with that of the induced state ( 1VQN ) ( Schmeing et al . , 2005b ) in the PTC region . Morphing between two models was shown . Critical residues of SecM and the 23S rRNA are labeled . As shown , a deformation of the CCA-end of the peptidyl-tRNA disrupts canonical P-loop interaction . Also see Figure 7 and text for details . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 024
In the present work , we obtained two high-resolution structures of SecM-arrested ribosomes , one in the post-translocational state and the other in the rotated state . The first one , SecM-Gly-RNC , contains a peptidyl-tRNA in classical P/P-site and therefore is expected to display an overall conformation similar to the classical PRE/POST states of the ribosome . However , compared with the cryo-EM solution structures of the pre-translocational ribosome ( three tRNAs ) ( Brilot et al . , 2013 ) or the 70S·EF-Tu complex ( Villa et al . , 2009 ) from E . coli , an apparent mode of motion for the 30S subunit could be identified , featuring a rotational movement of the 30S body domain along its long axis ( Figure 6A , B ) . Notably , the specific conformation of the post-state ribosome was previously captured in E . coli crystal structures of the 70S-EF-G/GMPPCP complex ( Pulk and Cate , 2013 ) . Moreover , compared with the E . coli crystal structure of the post-initiation ribosome ( with one P/P-tRNA ) ( Dunkle et al . , 2011 ) , similar movement of the 30S subunit is also evident ( Figure 6C , D ) . This movement could result in a displacement of the peripheral parts of the 30S subunit up to ∼5 Å distance , and increase the spacing between the two subunits to a certain extent . In contrast , the central part of the 30S subunit ( platform domain ) remains largely unchanged ( Figure 6C–F ) . Furthermore , similar rotational movement of the 30S body also prevails ( Figure 6—figure supplement 1 ) , when compared with Thermus thermophilus crystal structures of the ribosome in pre-translocational state ( Jenner et al . , 2010; Selmer et al . , 2006 ) , the post-translocationl state bound with EF-G ( Gao et al . , 2009 ) , or the 70S·EF-Tu complex ( Voorhees et al . , 2010 ) . Very interestingly , this observation is very similar to a recent finding that mammalian ribosomes possess a novel motion mode for the small subunit , named ‘subunit rolling’ , to distinguish its POST and PRE state , and to regulate the elongation cycle ( Budkevich et al . , 2014 ) . However , due to the lack of reference structures ( especially high-resolution cryo-EM solution structures of the ribosomes in the POST and PRE states ) , we were unable to determine whether the rolling is a consequence of the stalling , or a general feature intrinsic to the POST-state ribosome . Nevertheless , this suggests the ‘subunit-rolling’ is another common mode of motion for both prokaryotic and eukaryotic ribosomes . 10 . 7554/eLife . 09684 . 025Figure 6 . A rolling-like motion of the 30S subunit in SecM-Gly-RNC . ( A ) Comparison of the cryo-EM density map of the 30S subunit in SecM-Gly-RNC ( yellow ) with that of a pre-translocational state ribosome with three tRNA-bound ( EMDB 5796 , orchid ) ( Brilot et al . , 2013 ) from an intersubunit view ( left panel ) and a side view ( right panel ) . ( B ) Same as ( A ) , but compared with the 30S subunit from the cryo-EM density map of a 70S ribosome bound with elongation factor-Tu ( EF-Tu ) ( EMDB 5036 , orchid ) ( Villa et al . , 2009 ) . Alignments were done using the segmented 50S subunit as reference . ( C ) Comparison of the atomic model of the 30S subunit in SecM-Gly-RNC ( cyan ) with that of the crystal structure of a post-translocational/initiation state ribosome ( PDB ID 4V9D , blue ) ( Dunkle et al . , 2011 ) . A side view of two superimposed models is shown on the left , with a zoom-in view on the right . ( D ) Temperature map of the 30S subunit in SecM-Gly-RNC , compared with the 30S subunit in the crystal structure of a post-translocational/initiation state ribosome ( PDB ID 4V9D ) . The 30S subunit in SecM-Gly-RNC is colored according to its distance deviations from 4V9D , with coloring scheme showing on the right . ( E ) Same as ( C ) , but for the comparison between SecM-Gly-RNC ( cyan ) and the atomic model of the cryo-EM structure of a post-translocational ribosome bound with EF-Tu ( PDB ID 4V69 , blue ) ( Villa et al . , 2009 ) . ( F ) Same as ( D ) , but the temperature map is for the comparison in ( E ) . The alignment was done using the 23S rRNA residues 1600–2800 as reference . The RMS deviations between respective reference sequences are 1 . 7 Å for 4V9D vs SecM-Gly-RNC ( C , D ) , and 1 . 4 Å for 4V69 vs SecM-Gly-RNC ( E , F ) . EF-Tu: Elongation factor-Tu . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 02510 . 7554/eLife . 09684 . 026Figure 6—figure supplement 1 . The rolling-like motion of the 30S subunit in SecM-Gly-RNC , compared with crystal structures of various ribosomal complexes from Thermus thermophilus . ( A-D ) Comparison of the 30S subunit in SecM-Gly-RNC ( cyan ) with that from the crystal structures of different ribosomal complexes from T . thermophilus , including a pre-translocationalstate ( three tRNAs ) ( PDB ID 4V51 , blue ) ( Selmer et al . , 2006 ) ( A ) , another pre-translocationalstate ( three tRNAs ) ( PDB ID 4V6F , blue ) ( Jenner et al . , 2010 ) ( B ) , post-translocation state with EF-G ( PDB ID 4V5F , blue ) ( Gao et al . , 2009 ) ( C ) , and 70S complex with EF-Tu ( PDB ID 4V5L , blue ) ( Voorhees et al . , 2010 ) ( D ) . Comparisons are based on a common 50S alignment . The alignment was done using the 23S rRNA residues 1600–2900 as reference . The RMS deviations between respective reference sequences are 1 . 6 Å for 4V51 vs SecM-Gly-RNC ( A ) , 1 . 6 Å for 4V6F vs SecM-Gly-RNC ( B ) , 1 . 6 Å for 4V5F vs SecM-Gly-RNC ( C ) , and 1 . 7 Å for 4V5L vs SecM-Gly-RNC ( D ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 02610 . 7554/eLife . 09684 . 027Figure 6—figure supplement 2 . Rotation of the 30S subunit relative to the 50S subunit in SecM-Pro-RNC . ( A-D ) Temperature map of the 30S subunit in SecM-Pro-RNC , compared with the 30S subunits in unrotated ribosomes , including the post-translocational state from Thermus thermophilus ( PDB ID 4V6F ) ( Jenner et al . , 2010 ) ( A ) , the pre-translocational state from T . thermophilus ( PDB ID 1VY5 ) ( Polikanov et al . , 2014 ) ( B ) , the post-translocational state from Escherichia coli ( PDB ID 4V9D ) ( Dunkle et al . , 2011 ) ( C ) , and the atomic model of SecM-Gly-RNC ( D ) . The 30S subunit in SecM-Pro-RNC is colored according to its distance deviations from the other , with coloring scheme showing on the right . The alignment was done using the 23S rRNA residues 1600–2900 as reference . The RMS deviations between respective reference sequences are ∼1 . 6 Å for 4V6F vs SecM-Pro-RNC ( A ) , 1 . 5 Å for 1VY5 vs SecM-Pro-RNC ( B ) , 1 . 2 Å for 4V9D vs SecM-Pro-RNC ( C ) , and 1 . 0 Å for SecM-Gly-RNC vs SecM-Pro-RNC ( D ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 02710 . 7554/eLife . 09684 . 028Figure 6—figure supplement 3 . Map density of decoding centers in SecM-stalled RNCs . ( A ) The conformation of A1492 and A1493 in SecM-Gly-RNC . Map density is shown in mesh , and the atomic model in stick representation . P/P-tRNA , mRNA and 16S rRNA are colored red , green , and azure , respectively . ( B ) The conformation of A1492 and A1493 in SecM-Pro-RNC . Map density is shown in mesh , and the atomic model in stick representation . A/P*-tRNA , mRNA and 16S rRNA are colored forest green , green , and blue , respectively . ( C ) Map density of P/P-tRNA and mRNA in SecM-Gly-RNC . The anti-codons of P/P-tRNA contact with codons of mRNA at positions from + 1 to + 3 . Map density is shown in mesh , and the atomic model in stick representation . P/P-tRNA and mRNA are colored red and green , respectively . ( D ) Map density of A/P*-tRNA , P/E-tRNA , and mRNA in SecM-Pro-RNC . The anticodons of A/P*-tRNA and P/E-tRNA contact with codons of mRNA at positions from + 4 to + 6 and from + 1 to + 3 , respectively . The positions of nucleotide bases in mRNA were numbered by setting the codon of Gly as + 1 to + 3 . Map density is shown in mesh , and the atomic model in stick representation . A/P*-tRNA , P/E-tRNA and mRNA are colored forest green , yellow , and green , respectively . The density map is displayed at ∼4σ contour level . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 028 The other structure , SecM-Pro-RNC , contains a peptidyl-tRNA in A/P* site and a tRNA between P/E site and pe/E site , displaying a typical rotated conformation . Compared with the crystal structure of the hybrid-state ribosome ( Dunkle et al . , 2011 ) , or the cryo-EM structure of the pre-translocational state trapped with elongation factor-G and viomycin ( Brilot et al . , 2013 ) , the degree of 30S subunit rotation relatively to the 50S subunit , the configuration of the intersubunit bridges , and the interactions between tRNA , 16S rRNA and mRNA in the decoding center are all highly similar ( Figure 6—figure supplement 2 and Figure 6—figure supplement 3 ) . Therefore , the similarity of the overall conformations of SecM-Gly-RNC and SecM-Pro-RNC to known structures of functional ribosomal complexes ( both unrotated and rotated ) suggests that SecM-induced stalling is likely due to the localized conformational changes of the PTC , induced by extensive interactions between the tunnel components and the nascent chain . Early low-resolution cryo-EM data suggested that recognition of SecM by the tunnel induces a cascade of rRNA rearrangements , propagating from the exit tunnel throughout the large subunit to reorient small subunit rRNA elements , and directly lock the ribosome and mRNAtRNAs complex ( Mitra et al . , 2006 ) . This hypothesis was argued by improved cryo-EM structure of SecM-arrested ribosome ( SecM-Gly-RNC ) at 5 . 6 Å resolution ( Bhushan et al . , 2011 ) , which did not reveal any significant , systematic , and large-scale rRNA arrangement on the 50S subunit . Instead , it was proposed that an observed ∼2 Å shift in the position of the tRNA-nascent peptide linkage of SecM-tRNA away from the A-tRNA reduces the rate of peptide bond formation , resulting in translation stalling . However , with much improved resolution , we did not observe significant movement of the CCA-end of the P-site tRNA . Instead , the position of the tRNA-nascent peptide linkage of SecM-tRNA is similar to that seen in the structure of the ribosome with active PTC ( Figure 4B ) ( Polikanov et al . , 2014 ) . Through the comparison with reference states of the ribosomes that are capable or incapable of peptide-bond formation , we could provide an alternative mechanism of SecM-induced stalling in SecM-Gly-RNC ( Figure 7A ) . 10 . 7554/eLife . 09684 . 029Figure 7 . Schematic models of SecM-mediated translation stalling . ( A ) Model illustrating how interactions of SecM within the ribosomal tunnel in SecM-Gly-RNC promote conformational rearrangements of the 23S rRNA nucleotides of U2585 , U2506 , and A2602 , and thereby locks the PTC in the inactive state and not optimized for stable binding and accommodation of the incoming A-site tRNA . ( B ) Model illustrating how interactions of SecM within the ribosomal tunnel in SecM-Pro-RNC alter the interaction between the A/P*-tRNA and the P-loop . The altered P-loop interaction increases the life time of A/P*-tRNA and slows down its translocation to classical P/P site . DOI: http://dx . doi . org/10 . 7554/eLife . 09684 . 029 When the ribosome encounters the SecM arrest sequence , W155 and F150 are fixed at relatively narrow constrict sites in the middle tunnel region , which in turn causes the compaction of the C-terminal half of the peptide ( Woolhead et al . , 2006 ) . As a result , R163 is confined in a specific position surrounded by U2506/C2452/A2451 ( Figure 2D ) , leading to a strong interaction between U2585 and the O3 in A76 of CCA-end of the P-site tRNA . These altogether inactivate the PTC in an uninduced state . Particularly , positions of the U2585/U2506 and R163 would create a steric hindrance for the precise accommodation of the incoming aminoacyl-tRNA to the A-site . In addition , when an aminoacyl-tRNA , which should be proline-tRNA in the case of SecM , reaches the A-site of the PTC , the naturally slow peptide bond formation rate for proline ( Pavlov et al . , 2009 ) would further enhance the stalling . Indeed , the intersubunit FRET experiments showed that the lifetime of the unrotated ribosome would significantly increase when the codon of P166 is positioned at the A-site ( Tsai et al . , 2014 ) . Therefore , the inhibitive conformation of the PTC and low peptide bond formation rate of P166 collectively causes the stalling of the translation in SecM-Gly-RNC . As noted in previous cryo-EM study ( Bhushan et al . , 2011 ) , only a small fraction of ribosomes contains both A/A and P/P tRNAs in the sample of SecM-stalled ribosomes . We had similar observation ( Figure 1—figure supplement 3 ) . In our dataset , there is a minor population ( 13% ) of particles that resulted in a low-resolution map ( 8 . 6 Å ) of an unrotated RNC with two tRNAs , one in the A/A-site and the other in the P/P-site . This structure is likely a snapshot of the SecM-arrested ribosomes with a prolyl-tRNA at the A-site ( before peptide-bond formation ) . However , due to the resolution limitation , the nascent chain density of the P/P-tRNA is relatively weak and thus could not be quantitatively analyzed . Notably , a very recent study reported high-resolution structures of Bacillus subtilis ribosome arrest on MifM peptide ( Sohmen et al . , 2015 ) . Although SecM and MifM show completely no sequence homology , they appear to employ very similar mechanism to inactive the PTC into an uninduced state to cause stalling . This indicates that the stalling mechanism in SecM-Gly-RNC we reported here is highly conserved across species . In the present study , we also determined the structure of SecM-Pro-RNC in rotated conformation at 3 . 3 Å resolution . In the previous cryo-EM study of SecM-stalled ribosomes , a similar fraction of ribosomes in hybrid state was also observed . But , due to the lack of sufficient resolution , it was not explicitly discussed ( Bhushan et al . , 2011 ) . Our hypothesis of SecM-Pro-RNC being a stalled form is supported by previous biochemical and recent FRET data . First , using oligonucleotide probes that are highly specific for either tRNAGly , tRNAPro , or tRNAAla , it was found that stalling can occur at either G165 or P166 because there were additional bands detected for arrest sequences ( Muto et al . , 2006 ) , and the fraction of peptidyl-tRNAPro increased markedly when the SecM construct was truncated to end at P166 ( Muto et al . , 2006 ) . Second , the intersubunit FRET experiments ( Tsai et al . , 2014 ) show that the lifetime of the rotated-state ribosome significantly increased when the codon of P166 is at the A-site . Therefore , combining our structural observations in SecM-Pro-RNC , a stalling mechanism in SecM-Pro-RNC could be outlined in the below ( Figure 7B ) . Interactions between the nascent chain and the tunnel in SecM-Pro-RNC are considerably different at atomic level from those in SecM-Gly-RNC . At the middle region , W155 and F150 interact with different components of the tunnel ( Figure 3F , G and Figure 3—figure supplement 1D ) ; however , the movements of W155 and F150 are smaller than the length between two Cα atoms of the mainchain , which leads to a further compaction of the C-terminal half of the SecM peptide compared with that in the SecM-Gly-RNC . The more compact conformation of the C-terminal half alters the interaction network between SecM and the ribosome as exemplified by the extensive interactions between R163 , I162 , Q158 , and Q160 with the tunnel ( Figure 4D , E and Figure 3—figure supplement 1B , C ) . These interactions do not significantly change the conformation of the ribosome , but lead to a distortion on the CCA-end of the A/P*-tRNA and thereby disrupting the canonical base paring between the CCA-end and the ribosomal P-loop . As shown in previous FRET data , during translation of SecM peptide , the lifetime of the rotated state of the ribosome significantly increases between codons , requiring increased EF-G sampling without inhibiting EF-G binding ( Tsai et al . , 2014 ) . Therefore , the impaired accommodation of the A/P-tRNA into the ribosomal P-loop might be responsible for observed delay in tRNA translocation from hybrid A/P site into classical P/P site in the FRET data ( Figure 3B and Figure 3—figure supplement 1 ) . This indicates , in addition to its inhibitive effect on peptide bond formation , SecM also slows down tRNA translocation . It should be noted that , similar as previous studies of ribosome stalling ( Bhushan et al . , 2011; Frauenfeld et al . , 2011; Halic et al . , 2006; Yap and Bernstein , 2009 ) , chloramphenicol was added to maximally preserve the RNC during sample preparation . Chloramphenicol is known to inhibit translation by binding near the PTC ( Dunkle et al . , 2010 ) . In our structures , we could observed chloramphenicol density in the map of SecM-Pro-RNC , but not in SecM-Gly-RNC ( Figure 1—figure supplement 7 ) . Notably , this is in contrast to the crystal structure where chloramphenicol binds to unrotated ribosome ( Dunkle et al . , 2010 ) . Chloramphenicol is observed to interact with C2452/A2503/G2061 of the 23S rRNA in our structure ( Figure 1—figure supplement 7D ) , instead of C2452/G2505/A2062 in the crystal structure ( Dunkle et al . , 2010 ) . Additionally , critical residues of U2585 , U2506 , and A2602 in our structure show apparently different conformations from that in the crystal structure ( Figure 4—figure supplement 1 ) . Although we could not accurately estimate how much chloramphenicol has contributed to the ribosome stalling in SecM-Pro-RNC , it appears that it is not the determining factor , given its non-specific effects to the identity of translating peptides . In this work , our analysis of ribosome–SecM interactions at near-atomic level reveals two different mechanisms of stalling and provides rich details for how SecM nascent peptide could module the translation in distinct steps . Together with previous and recent structural studies of ribosomes stalled through different ways , including antibiotics ( for examples , see refs ( Jenner et al . , 2013; Nguyen et al . , 2014; Schluenzen et al . , 2006; Wilson , 2014 ) ) , leader peptides ( such as SecM , Erm , TnaC , MifM ) ( Arenz et al . , 2014a; Arenz et al . , 2014b; Bhushan et al . , 2011; Bhushan et al . , 2010; Bischoff et al . , 2014; Seidelt et al . , 2009; Sohmen et al . , 2015 ) , it shows that induced conformational changes of the ribosome by nascent peptide and/or small ligand could fine tune translation rate in various mechanisms . Especially , the mechanism of the PTC inactivation in SecM-Gly-RNC is highly similar to other systems of nascent peptide-mediated stalling , including TnaC ( Bischoff et al . , 2014 ) , MfiM ( Sohmen et al . , 2015 ) , ErmCL ( Arenz et al . , 2014a ) , and ErmBL ( Arenz et al . , 2014b ) , indicating that a few PTC residues such as A2062 , U2585 , U2586 , and A2602 are common responsive elements which take conformational signal from nascent chains within the tunnel to modulate the kinetics of translation elongation . This could have a deeper implication in translation control , as it highlights a means of the ribosome to adjust translation rate constantly in codon-specific manners when moving along mRNAs .
Two similar SecM constructs ( Figure 1—figure supplement 1A ) were generated by the overlapping PCR ( polymerase chain reaction ) . In the first step , the 2xStrep-TEV fragment was amplified using a pair of primers: a forward primer containing 2xStrep-TEV ( 5’-AAACATATGGCAAGTTGGAGC-3’ ) and a reverse one containing OmpA-TEV ( 5’-GAGAATCTATACTTCCAAGGTATGAAAAAGACAGCTATCGCG-3’ ) with the pASG-IBA vector ( IBA ) as the template . In the second step , the OmpA-Myc-SecM fragment was amplified using a pair of primers: a forward primer containing TEV-OmpA ( 5’-CAAGGTATGAAAAAGACAGCTATCGCGATTGCAGTG-3’ ) and a reverse one containing OmpA-Myc-SecM ( 5’-CTACCGTAGCGCAGGCCGAACAGAAACTGATCTCTGAAGAAGACCTGTTCAGCACGCCCGTCTGGATAAGCCAGGCGCAAGGCATCCGTGCTGGCCCTCAACGCCTCACCTAACTCGAGTTT-3’ ) with a previous pET-21b-OmpA construct ( Zhou et al . , 2012 ) as the template . For the amplification of the OmpA-Myc-SecM-6XHis fragment , the same forward primer and the template as for the OmpA-Myc-SecM fragment were used , while the reverse primer is changed to a primer containing OmpA-Myc-SecM-6XHis ( 5’- CTACCGTAGCGCAGGCCGAACAGAAACTGATCTCTGAAGAAGACCTGTTCAGCACGCCCGTCTGGATAAGCCAGGCGCAAGGCATCCGTGCTGGCCCTCAACGCCTCACCCTCGAGTTT-3’ ) . In the final step , the two fragments obtained above , 2xStrep-TEV and OmpA-Myc-SecM , were mixed to serve as the template , and a PCR was carried out with two primers 2xStrep-TEV and OmpA-Myc-SecM to amplify the 2xStrep-TEV-OmpA-Myc-SecM fragment . The 2xStrep-TEV-OmpA-Myc-SecM-6XHis fragment was similarly amplified . Both fragments were cloned into pET-21 vectors . Constructs were confirmed by DNA sequencing . SecM-RNCs were generated using an E . coli in vitro S30-T7 high-yield protein translation system ( Promega Corporation , Madison , WI ) according to the manufacturer’s instruction . The RNCs were purified as previously described ( Bhushan et al . , 2011 ) with modifications . Briefly , a 500-μl reaction was incubated at 30°C for 15 min on a shaker , and then spun through 500 μl sucrose cushion ( 50 mM HEPES , 250 mM KOAc , 25 mM Mg[OAc]2 , 5 mM DTT , 750 mM sucrose , 0 . 1% Nikkol , 500 μg/ml chloramphenicol , 0 . 2 U/ml RNasin [Promega Corporation ] and 0 . 1% pill/ml Complete EDTA-free Protease inhibitor cocktail [Roche , Indianapolis , IN] , pH 7 . 0 ) at 35 , 500 g for 60 min in a TLA120 . 1 rotor ( Beckman Coulter , Brea , CA ) at 4°C . The pellet was resuspended in 1000 μl of ice-cold 250 buffer ( 50 mM HEPES , 250 mM KOAc , 25 mM Mg[OAc]2 , 5mM DTT , 250 mM sucrose , 0 . 1% Nikkil , 500 μg/ml chloramphenicol , 0 . 2 U/ml RNasin , 0 . 1% pill/ml Complete EDTA-free Protease inhibitor cocktail , pH 7 . 0 ) for 60 min at 4 °C , then transferred onto 500 μl of Strep Affinity Resin ( IBA ) and incubated for 2 hr at 4°C . The resin was then washed with 5 ml of ice-cold 250 buffer and 2 ml 500 buffer ( 250 buffer with 500 mM KOAc ) . RNCs were eluted with 1 . 2 ml of 250 buffer containing 0 . 5 mM desthiobiotin . Each 600 μl eluted RNCs were spun through 400 μl of a high-salt sucrose cushion at 55 , 000 r . p . m . for 4 hr in a TLA 55 rotor ( Beckman Coulter ) at 4°C . The RNC pellet was resuspended in 150 μl of grid buffer ( 20 mM HEPES , 50 mM KOAc , 6 mM Mg[OAc]2 , 1 mM DTT , 500 μg/ml chloramphenicol , 0 . 05% Nikkol , 0 . 5% pill/ml Complete EDTA-free Protease inhibitor cocktail , 0 . 1 U/ml RNasin , and 125 mM sucrose , pH 7 . 0 ) . Initial attempts to image the RNCs obtained above by cryo-EM were hindered due to the presence of some polysomes . Therefore , improved sample purification was achieved by adding 20 U of RNaseA ( incubated for less than 5 min ) into the reaction before the first spinning through sucrose cushion to convert polysomes into monosomes . As a result , polysomes in SecM-stalled RNCs decreased dramatically and ribosomes enriched with strep-affinity resin remained intact as confirmed with cryo-EM . NuPAGE ( Life Technologies , Thermo Fisher Scientific , Waltham , MA ) and Western blotting confirmed that the peptidyl-tRNA was a single band . Purified SecM-RNCs were aliquoted in small volumes , flash frozen in liquid nitrogen , and stored at -80°C . To analyze the compositions of SecM-stalled RNCs , nascent peptides were purified from SecM-stalled RNCs . The SecM-stalled RNCs were first incubated in grid buffer with 10 mM EDTA and 5% pill/ml Complete EDTA-free Protease inhibitor cocktail to disassemble ribosomes for 30 min at 4°C , then treated with 100 U of RNase A to digest all forms of RNA for another 30 min at 4°C . The mixture was transferred onto Strep Affinity Resin ( IBA ) and incubated for 1 hr at 4°C . The resin was then washed with ice-cold 250 buffer and 500 buffer ( both with 10 mM EDTA ) . Nascent peptides were eluted with 250 buffer containing 0 . 5 mM desthiobiotin ( with 10 mM EDTA ) . Purified nascent peptides were analyzed by NuPAGE and Western blotting with primary antibody against Myc ( Cell signaling Technology , Danvers , MA ) or Strep ( Abcam , Cambridge , MA ) . In-gel digestion of the nascent peptide by lysyl Endopeptidase was carried out following the manufacturer protocol ( #125-05061 , Wako Pure Chemical Industries , Ltd . , Osaka , Japan ) . For LC-MS/MS analysis , digestion products were separated by a 60-min gradient elution at a flow rate of 0 . 300 µL/min with the EASY-nLC 1000 system , which was directly interfaced with a Thermo Orbitrap Fusion mass spectrometer ( Thermo Fisher Scientific , Waltham , MA ) . Mobile phase A consists of 0 . 1% formic acid and mobile phase B consist of 100% acetonitrile and 0 . 1% formic acid . The Orbitrap Fusion mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 3 . 0 software , and there was a single full-scan mass spectrum in the Orbitrap ( 350–1550 m/z , 120 , 000 resolution ) followed by top-speed MS/MS scans in the Ion-trap . The MS/MS spectra from each LC-MS/MS run were searched against the selected database using Proteome Discovery searching algorithm ( version 1 . 4 ) . To correct for beam-induced movements , the 14 movie frames ( 2–15 ) for each micrograph were aligned using the algorithm developed by Li et al ( Li et al . , 2013 ) . The swarm tool in the e2boxer . py program of EMAN2 ( Tang et al . , 2007 ) was used for semiautomatic picking of 238 , 212 particles from 3 , 908 micrographs . Contrast transfer function parameters were estimated using CTFFIND3 ( Mindell and Grigorieff , 2003 ) , and all 2D and 3D classifications and refinements were performed using RELION ( Scheres , 2012 ) . We used reference-free two-dimensional class averaging and three-dimensional classification to discard bad particles and 192 , 122 particles were selected for further three-dimensional refinement ( Supplementary file 1 ) . A previous cryo-EM map of the empty 70S ribosome was low-pass filtered to 70 Å and used as the reference for the following 3D classification . In the initial 3D classification , particles were split into six groups using an angular sampling of 7 . 5° . Two major classes were separated based on the large conformational difference of the 30S subunit and positions of tRNAs ( Figure 1—figure supplement 3 ) . Particles from these two major classes were combined and subjected to further 3D classification ( five classes ) with a final angular sampling of 1 . 8° . Three major classes were separated and showed improved resolution for the 70S ribosome with clear densities of nascent peptide chains in the exit tunnel . As a result , two of them were combined to yield a subset of 60 , 354 particles for SecM-Gly-RNC , and the other one yielded 41 , 501 particles for SecM-Pro-RNC . To improve the density quality of the nascent peptide chain , a soft mask of the 50S subunit ( plus tRNA ) was applied during structural refinement , along with an angular sampling of 0 . 9° combined with local angular searches around the refined orientations ( Figure 1—figure supplement 3 ) . Soft masks were made by converting atomic models into density maps , and adding cosine-shaped edges . Reported resolutions were based on the gold standard FSC = 0 . 143 criterion , and FSC curves ( Figure 1—figure supplement 4 ) were corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution ( Chen et al . , 2013 ) . The resulting density maps were corrected for the modulation transfer function of the detector and sharpened using postprocessing options of RELION 1 . 3 . Modeling of the structures of SecM-stalled RNCs was based on the crystal structure of the E . coli 70S ribosome ( PDB ID 4V7T ) ( Dunkle et al . , 2010 ) . First , the crystal structure was docked by rigid body fitting into the Cryo-EM density maps of SecM-Gly-RNC and SecM-Pro-RNC using EMfit ( Rossmann et al . , 2001 ) and UCSF Chimera ( Pettersen et al . , 2004 ) . Nascent chains were manually built using COOT . The overall fitting of the crystal structure showed an excellent agreement with our map densities . Then minor adjustments of the side chains of uL4 and uL22 , and nucleotides of 16S rRNA , 23S rRNA , and tRNAs , were manually performed using COOT ( Emsley et al . , 2010 ) . Next , the models were further refined against the density maps with stereochemical and secondary structure restraints using Phenix . real_space_refine ( Adams et al . , 2010 ) . At last , the refined models were subject to REFMAC ( Murshudov et al . , 1997 ) for further refinement in Fourier space , according to methods previously described ( Amunts et al . , 2014; Fernandez et al . , 2014 ) . To avoid overfitting , different weights for refinement were tested . For refinement of the atomic models of the 50S subunit ( 50S + tRNA + mRNA + SecM ) , the models were refined against the 50S-masked maps . After refinement , models of the 50S half were combined with fitted the 30S half to interpret the 70S maps . Final models were evaluated using MolProbity ( Chen et al . , 2010 ) , and statistics of reconstruction and model refinement were provided in Supplementary file 1 . For cross-validation against overfitting , we followed the procedure previously described ( Fernandez et al . , 2014 ) . This procedure involved the use of both ‘half maps’ that were calculated from the same halves of the particles as used for the gold-standard FSC calculations . To remove potential model bias from the final model that was built based on the density map from all particles , the atoms coordinates were displaced randomly by up to a maximum of 0 . 5 Å using PHENIX . This displaced model was then refined against one of the half maps using REFMAC with secondary structure , base pair and planarity restraints applied . FSC curves were calculated between the resulting model and the map it was refined against ( FSCwork , red curves in Figure 1—figure supplement 8 ) , and between the resulting model and the other half map ( FSCtest , blue curves in Figure 1—figure supplement 8 ) . The small separation between work and test FSC curves suggested that the models were not overfitted . Structural analysis and figure preparation were done with Pymol or Chimera . The density maps of SecM-Gly-RNC and SecM-Pro-RNC ( with 50S-based mask applied during refinement ) have been deposited in the EMData Bank under accession code of 6483 and 6486 , respectively , and their associated atomic models have been deposited in the Protein Data Bank under accession code of 3JBU and 3JBV , respectively . The density maps ( without 50S-based mask ) of SecM-Gly-RNC and SecM-Pro-RNC have also been deposited in the EMData Bank under accession code of 6484 and 6485 , respectively .
Regulation of translating ribosomes is a major component of gene expression control network . In Escherichia coli , ribosome stalling by the C-terminal arrest sequence of SecM regulates the SecA-dependent secretion pathway . Previous studies reported many residues of SecM peptide and ribosome exit tunnel are critical for stalling . However , the underlying molecular mechanism is still not clear at the atomic level . Here , we present two cryo-EM structures of the SecM-stalled ribosomes at 3 . 3–3 . 7 Å resolution , which reveal two different stalling mechanisms at distinct elongation steps of the translation cycle: one is due to the inactivation of ribosomal peptidyl-transferase center which inhibits peptide bond formation with the incoming prolyl-tRNA; the other is the prolonged residence of the peptidyl-RNA at the hybrid A/P site which inhibits the full-scale tRNA translocation . These results demonstrate an elegant control of translation cycle by regulatory peptides through a continuous , dynamic reshaping of the functional center of the ribosome .
Many genes code for proteins that carry out essential tasks . The instructions in a gene are first copied into a messenger RNA ( mRNA ) , and a molecular machine known as a ribosome reads the copied instructions in groups of three letters at a time ( called codons ) . The ribosome translates the order of the codons into a sequence of amino acids; each amino acid is carried into the ribosome by a transfer RNA ( tRNA ) molecule . As it translates , the ribosome joins each new amino acid to the one before it , like the links in a chain . Finally , the newly built protein chain passes through a tunnel to exit the ribosome . Ribosomes do not build all proteins at a constant rate; there are many examples of proteins that stall when they are in the ribosome exit tunnel . It is thought that this stalling is an important way for cells to control the expression of proteins . SecM is a bacterial protein that stalls while it is being made . Previous research has shown that a sequence of amino acids in SecM ( called the arrest sequence ) interacts with components of the ribosome tunnel . This interaction leads to stalling , and regulates the translation of another important bacterial protein ( called SecA ) that is encoded downstream on the same mRNA as SecM . If SecM-induced stalling takes place , the translation of SecA actually increases . Nevertheless , it remains poorly understood how SecM stalls in the ribosome . Zhang et al . have now solved the structures of SecM proteins stalled inside ribosomes using a method called cryo-electron microscopy . This approach identified two different states of SecM present in the ribosome , which corresponded to two different stalling mechanisms . The addition of an amino acid to a growing protein occurs in stages . First , the tRNA that carries the amino acid to the ribosome and bind to it in a region known as the A-site . After this , the tRNA moves to the P-site where the attached amino acid is incorporated into the elongating protein chain . Zhang et al . observed that the arrest sequence of SecM and the ribosome tunnel interact extensively . These interactions are strong and alter the configuration of both the A-site and P-site of the ribosome . This has two major consequences for translation . First , the tRNA cannot be stably accommodated in the A-site and secondly , its passage to the P-site is slowed down . Both these mechanisms contribute to stalling . This study provides a detailed analysis of how the ribosome can adjust to control translation . It also highlights that codon-specific control of translation constitutes an important component of how gene expression is regulated .