The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate. The bacterial RNA polymerase core enzyme is a multisubunit complex that synthesizes RNA using the DNA template as a guide. An additional subunit called sigma (σ) factor associates with the core enzyme and assists it in reading the signals in the DNA that tell it where to begin transcribing (Figure 6–11). Together, σ factor and core enzyme are known as the RNA polymerase holoenzyme; this complex adheres only weakly to bacterial DNA when the two collide, and a holoenzyme typically slides rapidly along the long DNA molecule and then dissociates. However, when the polymerase holoenzyme slides into a special sequence of nucleotides indicating the starting point for RNA synthesis called a promoter, the polymerase binds tightly, because its σ factor makes specific contacts with the edges of bases exposed on the outside of the DNA double helix (step 1 in Figure 6–11A).
The tightly bound RNA polymerase holoenzyme at a promoter opens up the double helix to expose a short stretch of nucleotides on each strand (step 2 in Figure 6–11A). The region of unpaired DNA (about 10 nucleotides) is called the transcription bubble and it is stabilized by the binding of σ factor to the unpaired bases on one of the exposed strands. The other exposed DNA strand then acts as a template for complementary base-pairing with incoming ribonucleotides, two of which are joined together by the polymerase to begin an RNA chain (step 3 in Figure 6–11A). The first ten or so nucleotides of RNA are synthesized using a “scrunching” mechanism, in which RNA polymerase remains bound to the promoter and pulls the upstream DNA into its active site, thereby expanding the transcription bubble. This process creates considerable stress and the short RNAs are often released, thereby relieving the stress and forcing the polymerase, which remains in place, to begin synthesis over again. Eventually this process of abortive initiation is overcome and the stress generated by scrunching helps the core enzyme to break free of its interactions with the promoter DNA (step 4 in Figure 6–11A) and discard the σ factor (step 5 in Figure 6–11A). At this point, the polymerase begins to move down the DNA, synthesizing RNA, in a stepwise fashion: the polymerase moves forward one base pair for every nucleotide added. During this process, the transcription bubble continually expands at the front of the polymerase and contracts at its rear. Chain elongation continues (at a speed of approximately 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal, the terminator (step 6 in Figure 6–11A), where the polymerase halts and releases both the newly made RNA molecule and the DNA template (step 7 in Figure 6–11A). The free polymerase core enzyme then reassociates with a free σ factor to form a holoenzyme that can begin the process of transcription again (step 8 in Figure 6–11A).
The process of transcription initiation is complicated and requires that the RNA polymerase holoenzyme and the DNA undergo a series of conformational changes. We can view these changes as opening up and positioning the DNA in the active site followed by a successive tightening of the enzyme around the DNA and RNA to ensure that it does not dissociate before it has finished transcribing a gene. If an RNA polymerase does dissociate prematurely, it must start over again at the promoter.