There have been more motorcycles in movies than we can count. But only a handful of the scenes have been so epic that we can't ever forget them.
Some are from old Hollywood classics while others are from modern day blockbusters. There are the ones most memorable for breakneck action sequences. And then there are the ones that are just plain funny.
Despite the best efforts of your mom, you've fallen in love with motorcycles. And you're certainly not alone: there are over 8.4 million motorcycles registered in America alone.
It's not just the riding that draws people in. It's the entire culture. From the history to restoring classic bikes to old-school biker gangs to the leather jackets and more.
There are few things in life more exhilarating than scenic motorcycle trips. The call of the open road is hard to ignore. As the seasons begin to change and the colors of autumn arrive, there's no better time to break out the map in search of an exciting destination.
If you love fall scenery, you are probably looking for the best routes for motorcycle trips this time of year. Let's take a look at a few that you won't want to pass up.
It's hard to beat motorcycle trips where you can soak in the gorgeous colors of the fall foliage. Here are some of the best destinations for autumn rides.
Beartooth Pass starts in the town of Red Lodge, Montana and takes motorcyclists on a journey across 68 miles into Wyoming and the northeast entrance of Yellowstone National Park. This is one of the most popular stretches of paved road in the Rocky Mountains among motorcycle enthusiasts. It offers stunning vistas and steep switchbacks, glacier-laden peaks, waterfalls, and expansive forests. This twisting, beautiful journey through the Rockies takes riders up to 10, 974 feet, and should definitely be added to every rider's top 10 list.
The northwestern section of Arkansas is known by many riders as the mother load of awesome motorcycle roads. At the top of the heap is a 19-mile stretch of pavement called Pig Trail. It follows Arkansas 23 and passes through a twisting tunnel of foliage in the Ozark and Boston Mountains. Among the highlights of the Pig Trail is the charming Victorian town of Eureka Springs, with tons of shops, restaurants, and lodging to take advantage of. Riders love the exhilarating hairpin turns and breath-taking scenery, so we recommend that you head to the Ozarks sooner rather than later.
This ride is only 11 miles long, but features 318 curves. It's a stretch of U.S. Highway 129 that borders Great Smoky Mountains National Park in Tennessee before dipping into North Carolina. Although it's the shortest ride on this list, its thrilling curves with names like Wheelie Hell, Brake or Bust Bend, and Copperhead Corner will challenge your riding skills and the fall colors will inspire you.
This iconic ride along the spine of the Blue Ridge and Appalachian Mountains actually connects Great Smoky Mountain National Park in North Carolina with Shenandoah National Park in Virginia. It's a long 469-mile ride that is often referred to as "America's Favorite Drive". It boasts amazing views with plenty of places to stop, along with a diverse variety of wildlife and foliage to enjoy as you motor along in the laid-back atmosphere.
Often referred to as Twisted Sisters, this 100-mile loop through the Texas Hill country might just change your preconceptions of the Lone Star State. Three Sisters takes riders up jagged hills, through canyons, and provides ample wide open views from multiple vistas. This ride is best taken during autumn when the temperature is cooler and the trees have begun to change colors. This is as good as it gets, with clear rivers and twisty mountain curves, allowing you to experience Texas at its most beautiful. Just be aware that there aren't many places to stop so you'll need to gas up before hitting the road.
PCH is really the quintessential American road trip. If this dream-like ride isn't already on your bucket list, add it immediately. It stretches for 655 miles from Seattle to San Diego, California, and is truly some of the most beautiful scenery in the country. Autumn is the ultimate time to experience this ride, as the winding pavement takes you through forests ablaze with vibrant red and orange of changing leaves. You certainly don't want to miss the 123 mile stretch of PCH between Monterey and Morro Bay, where riders will pass through Big Sur and Carmel-by-the-Sea, as well as the historic Hearst Castle. With the sun on your back and a warm breeze in your face, it's hard to beat a trip down the PCH.
If you're looking for a ride that offers the ultimate scenery, the Colorado Rocky Mountains rank second-to-none, and the San Juan Mountain Skyway is considered the best of the best. This circular 236-mile path will take you through the southwestern Colorado mountain towns of Telluride, Ouray, Silverton, and Durango. The road climbs to an elevation of 14,000 feet, passing through red rock canyons, hot springs, and historic mining towns, with inspiring mountain views along the entire route. Mesa Verde National Park and Rocky Mountain National Park are especially glorious as the colors of fall arrive.
Located in the western portion of North Carolina, the Cherohala Skyway extends all the way into remote parts of Eastern Tennessee. This route doesn't offer the extreme twists and turns of the nearby Tail of the Dragon. But considering that it's four times longer, it's actually preferred by most riders planning a trip in North Carolina. This easy ride is 45 miles long, making it a joy to see during the beautiful autumn months.
This one takes us through Wisconsin. Kettle Moraine Scenic Drive curves through sloping hills, steep ridges, and beautiful lakes. Kettle Moraine's incredible views were created by glacial movement millions of years ago. The route boasts amazing foliage in fall, and the plentiful variety of restaurants and shops we give you tons to do as you enjoy the brilliant colors.
Built in the 1930s, this route unfurls for 444 miles from Nashville, Tennessee to Natchez, Mississippi. Before becoming the Natchez Trace Parkway, it served as a migratory trail for Native Americans and buffalo. The ride will take you past Civil War battle sites and ghost towns, as well as parts of the original Natchez Trace Indian Trail. Don't worry about stops signs (there aren't any) or trucks (they are not allowed) as you make the journey from state to state.
This ride might be a mere 60 miles long, but it combines plenty of sensational twists with some of the most astounding scenery you'll find on the planet. The mountain vistas will dazzle you as the road crests the Continental Divide in the heart of Rocky Mountain National Park. The valleys are glacier-cut and the switchbacks meander through thick forests to alpine meadows ablaze with colors from a Monet painting.
Peak to Peak extends north along Highways 72 and 7, then drops down to Estes Park. There's plenty of wildlife to see there, including elk and moose. So keep to a safe speed, because hitting one of these huge animals will definitely ruin your day. We suggest packing your hiking books so that you can take a break from riding and walk a scenic trail inside the park. Or even take along some camping gear for weekend motorcycle trips.
U.S. Route 1 in Maine, from Brunswick to Machias is a quintessential New England experience. It's 167 miles of paved two-lane that winds through picturesque seaside villages and a landscape dotted with historic lighthouses. The road features both sweeping curves and long open sections. Just beware of moose and other wildlife that can appear out of nowhere.
This motorcycle paradise is ranked among the Midwest's most scenic roads. Located in Michigan, The Tunnel of Trees is part of the M-119, and winds under a canopy of leafy trees that burst into incredible colors in the fall. It starts in Harbor Springs and runs for 20 miles along a spectacular bluff that overlooks Lake Michigan, and features some especially sharp twists at Horseshoe Curve and Devil's Elbow. Be sure to treat yourself to the unique food and shops along the way.
This highway is also known as the Pacific Coast Scenic Byway. It treats riders to a scenic tour of Washington's Olympic Peninsula. The ride from Olympia to the Oregon border is about 350 miles, and from there the road continues down the Pacific coast. You'll get to enjoy the colors of the season as well as the colorful ocean tide pools, ancient forests, and alpine meadows, all part of the region's diverse ecosystem.
Autumn is the time of year when Mother Nature is at her best. The experience of motorcycle trips in the fall is like no other. As temperatures get cooler and colors change, you'll be inspired to pack your gear and head out to the best places to ride motorcycles.
We encourage you to take advantage of the beauty of the autumn and set out on the road to adventure. No matter where on this list you choose to explore, you'll be making memories with family and friends that will last a lifetime. Never been on a big ride before? We've got you covered with 10 tips for your first cross country motorcycle trip.
Motorcycle movies have been on the scene for over half a century. They range from films about biker gangs full of hardened and violent criminals to films about 1960s counterculture and Americana.
If you can't be on your motorcycle all of the time, why not take in a classic motorcycle movie. We've compiled a list of the top 13 motorcycle movies of all time to help you jump-start your biker film education.
There's nothing like the feel of the wind on your face while driving down the road in the summertime. But better yet is the view when you're riding down a scenic drive, be it coast or inland. Riders can take all their favorite accessories around the world and explore long and short trails alike, challenging their riding skills and taking in spectacular views. It's hard to know where to start when looking for a new drive to take, but this list encompasses some of the best drives all over the world.
Historically, motorcycles do great on tours, and adventure bike rentals have gone up in the last few years. It's important for a rider to be prepared for international travel. If a rider wants to take an international trip with their motorcycle, it's a good idea to have an international driver's license. There is more information here about how to obtain one. Several states, including Maine, have laws prohibiting wheelies, or "raising wheel." It's a good idea to check international laws before embarking on a trip overseas with a bike.
Traveling down both coasts and inland by motorcycle enables the rider to truly see the island. These trips are fairly short, only taking a couple hours at most to reach the end, but short trips mean riders can lengthen their tour with fun stops along the way. To tour the eastern coast of Sardinia, start in Olbia and follow SS125 south toward Tortoli. With good pavement, the breathtaking views of the coast are interspersed with tiny villages and even a castle! Along the way, there is a town called Genna Silana to stop for coffee. Tourists will find some great hiking trails, too, if riders want to stretch their legs. To tour Central Sardinia, take SS 198 from Tortoli toward Lanusei. Keep following that road through Gairo Vecchia and Osini, then follow the signs for Scala di San Giorgio. The last part of the trip to the top of the mountain is a tough ride in a forest, but worth the five minutes of roughness for the view at the end.There are few places to stop along the way, so make sure to fill up and have a bite to eat before leaving Tortoli. To tour western Sardinia, take SP 105 from Alghero to Bosa. This part of the trail is only 28 miles, but there are huge cliffs and perfect views all along the way. The road is clean and clear of traffic most of the time, too.
One of the shorter trips on the list, traveling along 51 is about a 30-40 minute drive. The landscape is beautiful, and tourists will begin at what has been heralded as the most beautiful city in the world, San Miguel de Allende. When riders reach Dolores Hidalgo, the site of the beginning of Mexico's independence, they will have plenty of sightseeing opportunity. Visit the church, the museums, and the monument, and then have lunch or dinner and a siesta.
This trail is 187 miles long, with all the curves a motorcyclist could want. It's almost a complete circle along the coast of Cape Breton Island, starting at Buckwheat Corner and traveling west. The trail goes through tiny sea towns and fishing villages, looping north, east, and back south, finishing at St. Ann's. There are so many places to stop along the way, with Celtic music and heritage to explore and wonderful hiking trails. The ocean is never far from view, and the road is generally in good condition.
This trail curves from Melbourne to Peterborough and was built in the 1920s. The best way to travel the road is over several days, although the trip is possible in only one. The seacoast and amazing landscape are visible, including Shipwreck Coast, and there are many beaches where riders could take a break for surfing. Riders can view the limestone rock formations, and they should make sure to stop at a pub along the way.
In order to cover the 1050 miles on this circuit, the riders start north along the western coast of South Africa, before heading east. The loop back to Cape Town can be completed in one or two weeks, depending on how much riders want to tour the area. Take the trail all the way to Addo National Park, and find some great curves and turns along Route 62. Don't forget your camera, and pick up some South African wine as you head back to Cape Town. Riders will want to fit in some tourism when they get back to the city, including a hike up Table Mountain (take a cable car back down). For a taste of Irish cuisine, visit The Dubliner downtown, and visit the colorful houses in the Bo-Kaap neighborhood, along with the market.
This is a very short drive, but there are over 300 curves in the short distance (11 miles!) of trail. Riders will travel the border of the Great Smoky Mountains on US 129, starting in North Carolina and ending in Tennessee. The mountains offer scenic views and beautiful forests, and along the way, you can find the Tree of Shame, covered in smashed up parts from motorcycles that were damaged on their tours of the Tail of the Dragon.
In the 60 miles of road from the Kipahulu Forest in Hana to Kahului, there are over 600 turns. Experienced riders should also watch out for the 60-some bridges with only one lane, but the gorgeous views and greenery is all worth it. Hana is isolated and not very developed, so the tropical paradise mostly remains untouched. Along the way, stop at the Hasegawa General Store, built in 1910 and featured in 1961 in the song by Paul Weston.
This is one of the longest permanent race tracks (although one of the shorter routes listed here) in the world, known worldwide as The Green Hell. Riders travel 12.8 miles with no speed limit and one-way traffic. Riders can pay a toll and drive around Nurburg and the castle there. The route is difficult, but the mountain views along the way are worth it. Sounds like a blast!
Starting in La Paz around 12,000 feet, the road climbs to 15,000 feet before descending to Coroico. It was built in the 1930s and is one of the only ways to get from La Paz, one of the nation's capitals and the government seat, down to the foothills. Years ago, hundreds of people died on this trail every year because of the gravel and the hairpin turns cliff side without guardrails. This is what gave the treacherous road its nickname, "The Death Road." Since 2008, the road has been paved, making it less dangerous, but still nerve-wracking to twist along the mountain unprotected. Before leaving La Paz, riders can do some sightseeing at the many museums and cathedrals.
This is a section of one of 40 border crossings between Argentina and Chile, and probably the most dangerous. The Paso de los Libertadores is full of switchbacks down the mountain, and there is always a lot of traffic. It is wise to travel slowly and safely. That is where the pass gets its name; Los Caracoles means "the snails." Along the way, view the magnificent statue Cristo Redentor de los Andes, a statue erected to commemorate the peace finally reached between the two countries.
Traveling the 270 miles from Kendal to Whitney is a one-day trip, but riders could stretch this into a longer excursion with tourist stops. Enjoying a pint after a long day's ride at a pub in northern England sounds like the perfect way to finish the drive. Along the way, visit Hawes, a small market town and the home of the Wensleydale Creamery. Leeds is also nearby (with Leeds Castle), Ripon, and the Yorkshire Dales National Park.
These are some fantastic places to visit. Riders have a ready-made bucket list now, with the advice to enjoy them in the sunniest of weather. These roads take travelers around the world, with short and long trips, tons of curves and straightaways, and the best vistas the world has to offer. Ready for the best motorcycle rides in the world? Zoom, Zoom!


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An Overdue Report on the Colorado Libertarian Party Convention
Posted by Diana Hsieh on 15 July 2002 at 12:42 pm	Uncategorized
Back in May, I gave a speech to the Colorado Libertarian Party Convention on the philosophical underpinnings of capitalism. I wrote up some thoughts on the experience immediately afterwards, but just never got around to editing and posting them. So now I've done both…
First off, kudos to Ari Armstrong for putting together an excellent convention. Apart from the (apparently usual) Libertarian time delays, all went smoothly. Ah well, one minor gripe, of which Ari was aware: The microphone was attached to the podium, so speakers were forced to boringly stand in a single spot during their speeches. During my speech, all of my Toastmaster training was screaming in protest at my lack of movement. Unfortunately, the more mobile microphone was of fairly poor quality, so stuck we were.
I was pretty pleased with my speech, in that I wasn't just regurgitating the usual libertarian dogma. I argued that there are four philosophical ideas that serve as "pillars" of freedom of trade: reason, egoism, harmony of interests, and mind-body integration. Rejection of any of these pillars leads to a particular form of statism (respectively): the paternalistic state, the welfare state, the egalitarian state, and the theocratic state. People who deny any of these philosophical ideas see economic arguments about the prosperity of capitalism as irrelevant; only philosophical arguments can save the day. So, as I said, it wasn't just the usual libertarian blah-de-blah. (I might propose an expanded version of this speech for the TOC summer seminar in 2003.)
Before I arrived at the convention, my basic worry was that I was presuming too little, that these philosophical issues would be old hat to most people. But after a few casual conversations and a sampling of the other lectures, I began to worry that perhaps I was presuming too much, that these philosophical issues would be foreign and undigestible to many.
My flip-flop was largely the result of my total lack of contact with purely Libertarian Party folks. I'm no neophyte to the libertarian movement, but my associations have been almost entirely though think tanks like The Cato Institute. (In fact, my only prior LP event was the Colorado LP's annual banquet last fall.) So, up until very recently, any big-L Libertarians I knew were also either Objectivists or super-smart policy wonks. Of course, I had heard all the usual stuff about the suits and freaks of the LP. But I expected the vast majority of attendees to be at least fairly well-educated about the substance of libertarianism. But many more than expected seemed to be fairly clueless. (Don't get me wrong: I'm not looking to bash LP folks as a whole, as lots of people there were clearly smart and informed.)
Most noticeably, far too many people seemed to be more anti-government than pro-freedom. Perhaps the most popular speech I heard was Douglas Bruce talking about his multiple fights and lawsuits with the government. The immense delight of the audience, I think, came largely from his tweaking the big bad authority of government, rather than his actually accomplishing anything worthwhile in the fight for freedom. It all smacked too much of the glee of an adolescent rebellion against authority. (And really, I don't have anything against Bruce, despite his stupid comment about my talk being sponsored by Enron.)
(As a contrasting side note: Shortly after the convention, I read this excellent piece by Ari on the dangers of the big brotherish ideas promulgated in a popular anti-methamphetamine video. Unlike Bruce, whose presentation focused almost entirely on his personal fights with government officials, Ari's commentary was all about the facts. His unpleasant run-in with the Larry Blunt, the News4 anchor who contributed to the video, merely served as an amusing and puzzling epilogue. That's the way it should be done.)
Also noteworthy was the fact that ending the war on drugs seemed to be a top priority for a great many people. Let me rephrase that: gaining the freedom to get high seemed to be a top priority for a great many people. Now, I'm all for ending the drug war. People have the right to put whatever substances they want into their bodies. And the drug war, like prohibition before it, promotes crime and fosters the worst in government. But the drug war is hardly the worst violation of rights we suffer here in the US. So by focusing heavily on the drug issue, Libertarians come off as a bunch of druggies just looking for an easier high. Of course, I suspect that that's precisely what a great many of them are.
Overall, I have to admit that I left the convention underimpressed with a great many of the participants and with the Libertarian Party as a whole. But I am grateful for the invitation from Ari to speak. Perhaps my speech got a few people thinking about some deeper philosophical issues.
Update: Today, I would not speak to the Libertarian Party, nor to any libertarian group. For my reasons why, see the second half of my blog post Stinky Garbage on Islam and my husband's essay The Fable of the Cardiac Surgeon and the Organization of Health Practitioners or Why I Don't Support Libertarian Organizations.
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3 Pipelines That Offer Amazing Income Possibilities
Enterprise Products Partners has three pipelines under construction that will provide plenty of income to patient investors
Callum Turcan
(callumturcan)
I love America and am proud to see us getting closer and closer to energy independence, which is why I cover energy stocks. Twitter: @CallumTurcan
To grow its distribution by 25% over the past five years, midstream energy specialist Enterprise Products Partners (NYSE:EPD) completed 70 major projects at a cost of $10 billion, which was slightly under budget. Looking ahead, the company is building three pipelines that could provide massive income upside for investors.
Six and a half million feet of pipeline
The Marcellus and Utica shale plays have suffered from a lackluster amount of takeaway capacity. Enterprise Products Partners was up to the challenge, and decided to build and link 1,230 miles of pipelines extending from Appalachia to Houston. The ATEX Express pipeline is due to open this quarter; it is designed to carry 125,000 barrels of day of ethane from the Utica and Marcellus shales to the Gulf Coast refinery complex for use as feedstock in petrochemical production.
There is still a lot of additional cash flow that can be wrung out of this investment. The ATEX Express is already connected to two natural gas liquids fractionators, and management plans on connecting two more NGL fractionators to the pipeline over the next six months.
As more fractionation capacity is hooked up to the pipeline, demand for NGL transportation via the ATEX Express will increase. Enterprise Products Partners wants to eventually increase the transportation capacity of the pipeline to 265,000 bpd if there is suitable demand. Unit holders shouldn't be worried about demand, as Enterprise Products Partners sees roughly 100 years' worth of drilling inventory in the Utica and Marcellus shales, leaving a long runway of NGL output growth from the region over the next few decades.
Don't forget about Oklahoma
The ATEX Express will help alleviate the bottleneck up north, but the shale revolution is widespread and pipelines are needed across the board. At the oil hub in Cushing, OK, surging crude output has caused a glut and depressed domestic oil prices. To remove the glut (which has already been slightly reduced due to the lower part of the Keystone pipeline coming online), Enterprise Products Partners is working with Enbridge (NYSE:ENB) to expand the Seaway pipeline, which carries crude from Cushing to Freeport, TX.
Enbridge made a wonderful call in 2011 when it bought ConocoPhillips' 50% stake in the pipeline. Since then, the Seaway pipeline's flow has been reversed and its capacity increased from 150,000 bpd to 400,000. Even better, Enterprise Products Partners and Enbridge plan to more than double the capacity of the Seaway pipeline to 850,000 bpd by the second quarter of 2014. For both companies, the completion of the Seaway expansion will provide a very bullish short-term catalyst that investors will love.
Hugging the Gulf
The third major pipeline Enterprise Products Partners under construction is the Aegis ethane pipeline, which will deliver ethane from Beaufort, TX, to Louisiana. Enterprise Products Partners has to date received commitments to transport 200,000 bpd of ethane, with the hope of eventually expanding that to 425,000 (peak capacity). The first phase is scheduled to be completed by the third quarter of 2014, and the second by the second quarter of 2015.
Foolish final thoughts
Enterprise Products Partners owns all kinds of midstream assets, but these three pipelines in particular offer unit holders the best chance of distribution growth over the next few years. The past five years have been great, but the next half-decade will be even better. Investors looking for a solid 4% yield with upside should take a closer look at Enterprise Products Partners.
For Enbridge, partnering up with one of the largest players in the midstream space seems to have been the right move. As the Seaway pipeline keeps churning out more and more cash flow each year, expect Enbridge to continue increasing its dividend.
Enbridge Inc.
NYSE:ENB
Enterprise Products Partners L.P.
3 Pipelines That Offer Amazing Income Possibilities @themotleyfool #stocks $ENB $EPD Next Article


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text:
A Triple Helix within a Pseudoknot Is a Conserved and Essential Element of Telomerase RNA
Kinneret Shefer, Yogev Brown, Valentin Gorkovoy, Tamar Nussbaum, Nikolai B. Ulyanov, Yehuda Tzfati
Kinneret Shefer
Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram, 91904 Jerusalem, Israel
Yogev Brown
Valentin Gorkovoy
Tamar Nussbaum
Nikolai B. Ulyanov
Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158-2517
For correspondence: ulyanov@picasso.ucsf.edu tzfati@cc.huji.ac.il
Yehuda Tzfati
DOI: 10.1128/MCB.01826-06
Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A·U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C·C+ triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C·U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.
Telomerase, a ribonucleoprotein reverse transcriptase, makes up for losses caused by incomplete DNA replication and degradation, by adding species-specific, 5- to 26-nucleotide (nt) repeats onto the telomere termini (reviewed in reference 2). The telomerase complex contains an RNA subunit (TER) (TLC1 in Saccharomyces cerevisiae), a catalytic reverse transcriptase (TERT) (Est2 in S. cerevisiae), and several other protein components. Unlike other reverse transcriptases, telomerase specializes in repeatedly copying a short RNA template within its integral RNA component.
TERs are highly divergent, being conserved in sequence only among closely related species. Phylogenetic covariation was used to predict conserved secondary structures for evolutionarily close species of ciliates (14, 24), vertebrates (4), Kluyveromyces budding yeasts of the K. marxianus cluster (31, 32), and Saccharomyces sensu stricto (3, 7, 13, 37). Limited similarity in the general architecture was observed among these models, consisting of three long arms and a catalytic core domain (4, 13). Although nontemplate regions are essential for the assembly, regulation, and function of telomerase, their specific roles are still unclear (reviewed in reference 30). We hypothesized that important functional elements would exhibit better conservation in their tertiary structures, rather than in their secondary structures or sequences. Solving these tertiary structures may provide insights into their conserved functions.
Pseudoknot elements were found to be critical for telomerase function in ciliates (28), vertebrates (4), and Kluyveromyces (32). For S. cerevisiae, alternative pseudoknot models were suggested but experimental data failed to distinguish between them (3, 7, 10, 13). Interestingly, a TLC1 region, which includes the putative pseudoknot, is required for Est2 binding (3, 13, 15). However, the exact binding site is yet unknown.
To study the pseudoknot structure in detail, we introduced disruptive and compensatory mutations into the Kluyveromyces lactis TER gene (TER1), which were designed according to a preliminary secondary structure model (32). As described below, not all of the resulting phenotypes supported the model, suggesting the importance of tertiary interactions. These results guided us in three-dimensional (3D) modeling of the pseudoknot using the computer program miniCarlo (40). Interestingly, the modeling revealed a triple-helical stem within the pseudoknot as the only apparent structure consistent with the in vivo mutant phenotypes. We confirmed this prediction in vitro using short RNA constructs and showed by disrupting and compensatory mutations that the triple-helix formation is essential for the function of telomerase in vivo. A recently published nuclear magnetic resonance solution structure of the human telomerase pseudoknot revealed a similar triple-helix motif (29), making it the first structural motif conserved across the highly divergent yeast and vertebrate telomerase RNAs.
Yeast strains, genomic DNA preparation, and Southern hybridization.K. lactis deletion strain yJR27, based on the parental strain 7B520, was used for the CEN-ARS plasmid shuffling of wild-type (WT) and mutant TER genes as described previously (25). Genomic DNA was prepared as described previously (18) except for omitting the proteinase K treatment. Southern analysis was performed as described previously (5). For BclI-specific hybridization, the 5′-end-32P-labeled oligonucleotide KTElBcl (GATCAGGTATGTGG, where the underlined base is a locked nucleotide analog at the position corresponding to the BclI mutation) was used as a probe, hybridized at 40°C, and washed for 5 min with 0.2 M Na+ twice at room temperature and once at 40°C. Hybridization with the WT telomeric probe (Klac1-25, ACGGATTTGATTAGGTATGTGGTGT) and the last wash were done at 50°C. Blots were exposed to film.
In vitro transcription of oligoribonucleotides.For radioactively labeled oligoribonucleotides, 80 pmol each of the T7 promoter oligonucleotide (common to all RNA constructs) and the specific CS3 or CS4 template oligonucleotide were incubated with 0.5 mM (each) CTP, ATP, and GTP; 0.1 mM UTP; [α-32P]UTP (3,000 Ci/mmol; added amounts were calculated according to the number of uridines incorporated to yield equal specific activity for all RNA sequences), 1× transcription buffer (supplied by the manufacturer), and 20 units of T7 RNA polymerase (Ambion Inc.) in a 20-μl reaction mixture for 2 h at 37°C and then treated with 4 units of RNase-free DNase (Ambion Inc.) at 37°C for 15 min. Unlabeled RNA was transcribed with a MEGAshortscript T7 kit (Ambion Inc.) according to the manufacturer's instructions. The following T7 promoter (in lowercase) and WT template sequences were used; mutations were introduced to transcribe the mutant sequences described in Fig. 3A and B and 6A and B: T7 promoter oligonucleotide, taatacgactcactatagg; WT CS3, GGTTGGAAAAATCACTAAAAAGAAAggtatagtgagtcgtatta; and WT CS4, TTGAGGTAAATGATTTTATGggtatagtgagtcgtatta. All oligoribonucleotides were gel purified on a denaturing 8 M urea-15% polyacrylamide gel in 1× Tris-borate-EDTA; eluted from the gel slice twice in 300 μl of 0.5 M ammonium acetate, 1 mM EDTA, and 0.2% sodium dodecyl sulfate at 37°C for 2 h; precipitated with 5 μg glycogen, 0.3 M sodium acetate (pH 5.2), and 3 volumes of ethanol; resuspended in H2O; and further purified with Microspin G-25 Sephadex columns (Amersham Biosciences).
Nondenaturing polyacrylamide gel electrophoresis.CS3 and/or CS4 oligoribonucleotides in the amounts indicated in the legends to Fig. 3 and 6 were heat denatured at 90°C for 2 min and cooled down at a rate of 1.7°C/min to 4°C in 44 mM sodium phosphate buffer (pH 6.25), 22 mM NaCl, and 5 mM MgCl2 (for Fig. 3C) or in 10 mM MOPS (morpholinepropanesulfonic acid) buffer (pH 5 or 7 for Fig. 6C and D, respectively) and 5 mM MgCl2. Then, 6% glycerol (for Fig. 3C) or 15% Ficoll (for Fig. 6C and D) was added, and samples were loaded onto nondenaturing polyacrylamide gels (15% or 10% acrylamide, respectively, and 1:40 bisacrylamide-acrylamide) in 6.7 mM Tris acetate, 3 mM sodium acetate, and 5 mM MgCl2, (pH 6.25, 5, and 7 for the gels in Fig. 3C, 6C, and 6D, respectively). Gels were run at 4°C and 120 V for 18 h (for Fig. 3C), at 20°C and 250 V for 3 h (for Fig. 6C and D, right panels), or at 30°C and 250 V for 2 h (Fig. 6C and D, left panels). Results were visualized by autoradiography. Dried gels were exposed to a phosphorimager (Molecular Dynamics) or to film, and bands were quantified using the ImageJ program. The percentage of the dimeric form relative to the total (monomeric and dimeric) RNA was calculated.
Cloning and sequencing of telomeres.Individual telomeres were cloned by a ligation-mediated PCR method (31). The anchor oligonucleotide (CGTCGGCCGCGTCGTGACT) was phosphorylated at the 5′ end and had an inverted dT attached to the 3′ end by a 3′-3′ phosphodiester bond to allow ligation only to its phosphorylated 5′ end. The ligation reaction mixture (20 μl) contained 60 pmol anchor oligonucleotide, 0.5 μg genomic DNA, 50 mM Tris-HCl (pH 8), 10 mM MgCl2, 1 mM dithiothreitol, 12.5% polyethylene glycol 8000, 1 mM hexamine cobalt chloride, 20 μM ATP (freshly added), 10 μg/ml bovine serum albumin, and 10 units RNA ligase (New England Biolabs) and was incubated for 90 min at 37°C. Ligated genomic DNA was purified by SureClean (Bioline Inc). Telomere fragments were amplified by PCR, using a subtelomeric primer (GACCGGGCCCAGCAGGACCAAG) present internally to 11 out of the 12 K. lactis telomeres and a primer complementary to the anchor oligonucleotides (AGTCACGACGCGGCCGACG). PCR products were gel purified, digested with ApaI and EagI (introduced in the primers), cloned into a Bluescript SK vector, and sequenced with the M13 reverse primer (from the telomere end towards the subtelomere).
Thermal melting.RNA samples (1.0 to 2.0 μM each) were incubated in 10 mM MOPS buffer and 5 mM MgCl2 at pH 5, 6, or 7. UV absorption (260 nm) was monitored with a UV spectrophotometer (Carry 300) attached to a temperature-controlled water circulator. The samples were incubated at 85°C for 2 min, and then the temperature was decreased to 20°C, paused at 20°C for 2 min, and then increased back to 85°C at a rate of 0.8°C/min with a 4-s data point averaging. Melting temperature (Tm) values were estimated from the graph of the first derivative of the curve of absorption versus temperature by using the computer program Origin.
Model building and molecular mechanic calculations.Model building and energy minimization of the pseudoknot structures were carried out with the miniCarlo program (40) on a cluster of Linux computers. The miniCarlo program performs molecular mechanic calculations of nucleic acid molecules, using internal coordinates as internal variables. The set of internal coordinates was designed specifically for nucleic acid structures, and it includes generalized helical parameters defining the relative positions of nucleic bases in space; aromatic bases are treated as rigid bodies with idealized geometry. The conformations of backbones connecting adjacent bases are calculated with a specialized chain closure algorithm (38), which ensures that bond lengths and bond angles have standard values. The conformational energy is calculated in vacuo with a distance-dependent dielectric constant, using an empirical force field optimized for nucleic acids (22, 39). To take the shielding effect of counterions into account, the phosphate groups are assumed to be electroneutral. Because of the simple geometric meaning of the independent variables, i.e., the helical parameters, the miniCarlo program can be used as a modeling tool with the help of the interactive molecular graphics program MIDASPlus (9) as described previously (33). Individual domains of the structure (such as stems or loops) were calculated with miniCarlo and then interactively arranged in space using MIDASPlus. After that, the internal coordinates defining the structure were calculated with Fitparam (34) and fed back to miniCarlo for the next iteration of energy optimization. Molecular graphics was prepared with MIDASPlus. The atomic coordinates of calculated models are available from the authors upon request.
We previously identified a putative pseudoknot element in K. lactis telomerase RNA and showed that it is essential for telomerase function (32). The proposed structure was composed of two conserved sequences: CS3, forming stem 1 and a U-rich loop, and CS4, pairing with CS3 loop residues to form stem 2 (Fig. 1B). An unusual 3-nt bulge between stem 1 and stem 2 was expected to sharply distort the coaxial alignment of the two stems, which is normally observed in RNA pseudoknots (reviewed in reference 12). While the formation of stem 2 was validated experimentally (32), stem 1 has not been tested before and its formation was supported only by phylogenetic covariation among Kluyveromyces species (32). A similar pseudoknot was proposed for S. cerevisiae TLC1 (3, 7, 13). However, deleting the corresponding stem 1 had no apparent effect on telomere length in S. cerevisiae (3, 13). To analyze the pseudoknot structure in more detail, we designed mutations that disrupted, restored, or paired the predicted stems and single-stranded regions. We replaced the WT TER1 gene in K. lactis with mutant genes by using a plasmid-shuffling system described previously (25) and tested the effects on telomerase activity in vivo. An additional single-nucleotide mutation was introduced into the TER template, resulting in the incorporation of BclI restriction sites into the telomeric repeats. Otherwise, this mutation is apparently silent and thus can be used to mark the nascent repeats incorporated by the investigated telomerase and reveal its in vivo activity (25). BclI repeats are identified by Southern analysis of telomeric fragments hybridized to a BclI-specific telomeric probe (Fig. 1A and D), or by BclI restriction endonuclease digestion and hybridization to a WT probe (Fig. 1A and E). The overall telomere length is revealed by the WT hybridization to telomeric fragments undigested with BclI (Fig. 1E). In yeast, a delicate balance between positive and negative regulation of telomerase activity maintains telomeres at a constant length. A change of less than 50% in the amount or activity of telomerase can impair the balance and affect telomere length (20). Therefore, telomere length can be used as a sensitive in vivo assay for telomerase activity. The results are described below.
Mutational analysis of the pseudoknot element. (A) Schematic representation showing telomeric fragments digested with restriction endonucleases EcoRI or EcoRI plus BclI and hybridization probes (WT and BclI specific). (B and C) Secondary structure model of the K. lactis pseudoknot element (B), with the positions of the mutations introduced to test stem 1, bulge, and stem 2 (C) highlighted in gray. (D and E) Plasmids carrying ter1 genes with the template BclI mutation alone (WT-BclI), additional pseudoknot mutations, or an empty vector (Δter), as indicated above the lanes (D), were used to replace the WT TER1 gene encoded on a URA plasmid. Genomic DNA was prepared from the resulting K. lactis strains in their sixth passage and analyzed by a Southern blot hybridized first with a BclI-specific (D) and then with a WT (E) probe. Only portions of the gel are shown, which include a group of 7 out of the 12 K. lactis telomeres. (F) Typical colony phenotypes of K. lactis strains taken at their fourth passage. Impaired telomere maintenance is associated with rough colony appearance, as opposed to the smooth WT colonies.
Stem 1.Two sets of mutations were designed to test stem 1. Each set consisted of two disrupting mutations in either strand of stem 1 and a double mutation (the combination of the two) predicted to restore the base pairing (Fig. 1B and C). One set was composed of 2-nt mutations (S1.1, S1.1′, and S1.1+1′) and another set of 4-nt mutations (S1.2, S1.2′, and S1.2+2′). Out of four single mutations, S1.1′ and S1.2 caused severe telomere shortening and a rough colony phenotype, a hallmark of impaired telomere maintenance in K. lactis (Fig. 1D, E, and F and Table 1). S1.2′ caused moderate telomere shortening, which did not affect the colony phenotype. In the fourth mutation, S1.1, two guanines were substituted for adenines and could potentially form wobble pairs with their uridine counterparts. As expected, this mutation did not affect telomere length and colony phenotype. It is not clear why the severity of the disrupting mutation S1.2 was different from that of S1.2′. It is possible that the more severe effects of S1.1′ and S1.2 reflect tertiary interactions impaired by these mutations. Alternatively, new interactions may form by the mutated sequences, which are more detrimental for the pseudoknot structure and function than the destabilization of stem 1 by S1.2′. However, it is important that the compensatory mutations fully restored normal telomere length and colony phenotype, confirming that the formation of stem 1 is important for telomerase function in vivo.
Summary of mutations and their effect on telomere function in vivo
The bulge.Normally, the two stems of a pseudoknot are stacked in a continuous helix. A bulge of 1 to 2 nt between the stems would introduce a kink in the helix, which may have biological implications (12). A preliminary 3D model (not shown) predicted that a 3-nt bulge between the two stems in the K. lactis pseudoknot, as appears in the secondary structure model (Fig. 1B), would introduce a sharp kink in the structure. To test the conformation and importance of the bulged nucleotides, we designed mutations to eliminate the bulged nucleotides or to pair them with CS4 residues (Fig. 1B and C). Deleting the 3-nt predicted bulge completely abolished telomerase activity, as revealed by the lack of BclI repeat incorporation (Fig. 1D), the deregulated telomere length (Fig. 1E, compare ΔB to Δter) typical of the telomerase-independent alternative pathway for telomere maintenance (17), and the rough colony phenotype (Fig. 1F). Surprisingly however, 3-nt substitutions in either CS3 or CS4, designed to pair the bulged nucleotides and eliminate the putative kink in the structure, only slightly reduced telomere length and did not affect colony phenotype (Fig. 1D, E, and F, BtS1 and BtS1′). In contrast, the double mutation composed of BtS1 and BtS1′ together abolished telomerase function in vivo (Fig. 1D, E, and F, BtS1+1′). These results suggested that the nucleotides previously thought to form a bulge might actually be paired, enabling the coaxial alignment of the two stems. Therefore, the BtS1 and BtS1′ mutants, which maintained base-pairing, did not significantly affect the structure. In the double mutant BtS1+1′, the substituted AUG876-878 nucleotides of BtS1′ may interact with the CUU861-863 of the CS3 loop, effectively extending stem 1 instead of stem 2. This change abolished telomerase function, indicating that the junction between the two stems and possibly the CS3 loop nucleotides are critical. The possibility of the two stems stacked into a continuous helix was puzzling—how could a pseudoknot structure accommodate a 10- to 12-nt-long stem 2 (a full helical turn of an A-form double helix) connected at both ends by a short 6-nt loop (Fig. 1B)? This puzzle inspired further research as described below.
Stem 2.Mutations in one part of stem 2 (UCA964-966 · UGA869-871) (Fig. 1B and C), designed to disrupt stem 2, caused severe telomere shortening. Compensatory mutations, designed to restore this part of stem 2 with altered sequences, fully restored telomerase function and normal telomere length, consistent with the pseudoknot model (S2.6, S2.6′, and S2.6+6′ in Fig. 1; S2.1, S2.1′, and S2.1+1′ in reference 32; and Tables 1 and 2). Similar results were obtained for the corresponding part of stem 2 in S. cerevisiae TLC1 (see mutants tlc1-29, tlc1-30, and tlc1-31 in reference 3 and no. 20, 21, and 22 in reference 13).
Characterization of double and triple mutants of stem 2
Interestingly, S2.7 telomeres were not as short as S2.6 telomeres (Fig. 1D and E). Since the only difference between the two mutations is an additional change of U967 to C in S2.7, this observation suggested that C967 is paired with G868 in the S2.7 mutant, partially compensating for the disruption of two A·U pairs. Altogether, these observations suggested that in the S2.7 and S2.7+6′ mutants, and possibly also in the WT, G868 is paired, implying that stem 2 may span over 12 bp (including the last A867U968 pair).
When the other part of stem 2 (AAAA960-963·UUUU872-875) (Fig. 1B) was tested, again single mutations in either strand severely disrupted or completely abolished telomerase function (S2.0 and S2.0′ in Fig. 2; S2.2, S2.2′, S2.3, S2.3′, S2.4, and S2.4′ in reference 32; and Table 1). However, in contrast to the first part of stem 2, in this part the double compensatory mutations did not restore at all or only partially restored normal telomere length (S2.0+0′, S2.2+2′, S2.3+3′, and S2.4+4′ in Fig. 2; Table 2) (32). Interestingly, a stretch of three to five A·U base pairs is conserved in this part of stem 2 in the predicted pseudoknots of Kluyveromyces, Saccharomyces, and vertebrate TERs. Similar results were obtained with S. cerevisiae for this corresponding part of stem 2 (mutations 17, 18, and 19 in reference 13 and tlc1-43, tlc1-44, and tlc1-45 in reference 3). We next asked whether the inability of double mutations in the AAAA960-963·UUUU872-875 part of stem 2 to restore normal telomerase function was caused by impaired CS3-CS4 interaction or by additional, unknown RNA-RNA or protein-RNA interactions. We synthesized short oligoribonucleotides composed of WT and mutated CS3 (28-nt) and CS4 (22-nt) sequences and analyzed their interaction by native polyacrylamide gel electrophoresis (PAGE) (Fig. 3). WT CS3 and CS4 together formed low-mobility bands, presumably corresponding to CS3-CS4 dimers (Fig. 3C, WT). Dimer formation was dependent upon Mg2+ ions (not shown). Incorporating 3-nt mutations designed to disrupt stem 2 (S2.1, S2.1′, S2.3, and S2.3′), abolished dimer formation. As expected, the double compensatory mutation S2.1+1′ fully restored dimer formation. Strikingly however, the double mutation S2.3+3′ in the AAAA960-963· UUUU872-875 part of stem 2 only partially restored dimer formation, despite the expected stabilizing effect of replacing three A·U with three G·C base pairs. Double transversion mutations at these positions, introducing three U·A pairs (S2.2+2′) or three C·G pairs (S2.4+4′) (not shown) almost completely abolished dimer formation. Furthermore, a 3-nt mutation (L.c) in the loop region of CS3 also completely abolished dimer formation, although the residues predicted to form stem 2 were unchanged. These results suggested that tertiary interactions were required to stabilize the dimer formation. Such interactions may have been weakened by the double mutation S2.3+3′ and completely disrupted by the double mutations S2.2+2′ and S2.4+4′ and by the loop mutation L.c. Importantly, the formation of stable dimers in vitro correlated well with the ability of the corresponding sequences to support telomerase function in vivo: the S2.3+3′ mutant had a moderate effect on telomerase function, while the S2.2+2′, S2.4+4′, and L.c mutants completely abolished or severely impaired telomerase function (Fig. 2; Tables 1 and 2) (32).
Mutational analysis of the triple-helical stem. (A) Secondary structure model of the K. lactis pseudoknot element. Highlighted in gray are the positions of the mutations introduced into the TER1 gene to test the triple-helical stem. (B and C) The mutations are described (B) and indicated in gray on the small schematic representations of the pseudoknot above the lanes in panel C. (C and D) Genomic DNA was prepared from the various K. lactis strains in their sixth passage and analyzed with a Southern blot hybridized with Bcl-specific (C) and WT (D) probes, as described for Fig. 1. (E) Typical colony phenotypes of the strains at their fourth passage. (F) WT hybridization to telomere fragments of strains grown at 16°C (for six passages on plates and then in liquid medium) instead of at the normal temperature of 28 to 30°C.
Dimer formation by oligoribonucleotides. (A and B) Secondary structure model of the K. lactis pseudoknot element (A), with the positions of the mutations introduced to test stem 2 and the CS3 loop (B) highlighted in gray. The first G residue in CS4 (in lowercase) is not present in the WT sequence but was introduced to facilitate efficient transcription by T7 RNA polymerase. (C) The WT and mutated CS3 and CS4 oligoribonucleotides (0.01 pmol each) described in panels A and B, both radioactively labeled, were denatured, renatured, and analyzed by 15% nondenaturing PAGE at 4°C and pH 6.25. Single oligoribonucleotides are indicated by CS3 or CS4 and asterisks below the lanes. Lanes with mixtures of both CS3 and CS4 are labeled by the name (above) and number (below) of the mutations introduced. Note that the mutant CS4-S2.3 oligoribonucleotide migrated slower than the WT CS4, presumably because the mutated (but not the WT) CS4 alone can form a stable dimer. Since it comigrated with the longer CS3, only one band corresponding to the monomer form appears under S2.3 and S2.3+3′.
Altogether, the conservation of primary sequence among species, the inability of compensatory mutations to fully restore dimer formation in vitro and telomerase function in vivo, and the effect of mutations in the CS3 loop suggested that the AAAA960-963·UUUU872-875 part of stem 2 and the CS3 U-rich loop are involved in non-Watson-Crick tertiary interactions essential for the formation of a stable pseudoknot structure.
Modeling a pseudoknot with a triple-helical stem.We checked the feasibility of forming a pseudoknot structure by CS3 (nt 856 to 883) and CS4 (nt 960 to 968), i.e., whether the sizes of stem 1 (nt 856 to 860 and 879 to 883), stem 2 (nt 867 to 875 and 960 to 968), and the loop (nt 861 to 866) are compatible with a pseudoknot architecture. At first we assumed that the UCC linker (nt 876 to 878) between the two stems remained single stranded. The critical question was whether the length of the loop, 6 nt, would be sufficient to span the length of stem 2 (initially assumed to be 9 bp), connecting the ends of stem 1 (nt 860) and stem 2 (nt 867). Modeling with the miniCarlo program showed that the length of the loop was indeed more than sufficient to fit into a pseudoknot structure (not shown). As is common for pseudoknots with two coaxial stems (36), the loop crossed the major groove of stem 2. It was located outside of the groove and had an irregular conformation.
However, as described above, mutational analysis suggested that the UCC876-878 sequence, previously thought to be single stranded, is actually paired. First attempts to model the stem extension mutants (BtS1 and Bts2+2′) (see Table 1) showed that the loop (nt 861 to 866) became very strained and started to penetrate the major groove of stem 2, which caused multiple interatomic clashes (not shown). It became obvious that for this mutant sequence to form a pseudoknot, the loop must be located inside the major groove of stem 2. A possible way to prevent interatomic clashes and stabilize this structure is to form a triple-helical stem. Although such a major-groove triple helix was first discovered for synthetic RNA in 1957 (8), high-resolution structures were available only for DNA. Therefore, we modeled a regular poly(rU)-poly(rA)·poly(rU) triple helix using miniCarlo (Fig. 4A). As in DNA triple helices (11), bases from a poly(rU) strand and a poly(rA) strand form Watson-Crick pairs, while the third strand, poly(rU), is located in the major groove in an orientation parallel to the poly(rA) strand, and its bases form Hoogsteen-type hydrogen bonds with the adenines (Fig. 4C). The backbones of all three strands have conformations typical of the A-family of forms, including the C3′-endo sugar puckers. The duplex formed by the Watson-Crick strands has a major groove somewhat wider than a typical duplex A-RNA, to accommodate the third strand. One distinction of the RNA triple helix from its DNA counterpart is an additional hydrogen bond between the hydroxyl group of each uridine of the third strand and the phosphate oxygen of the poly(rA) strand. This hydrogen bond is relatively weak, with a proton-to-oxygen distance of 2.4 Å, but it is nevertheless a stabilizing interaction. Using this triple helix, we built and refined the model for the stem extension BtS1 mutant (not shown). It has five consecutive U-A·U triples, U876-a959·U862, U875-A960·U863, U874-A961·U864, U873-A962·U865, and U872-A963·U866, incorporated in stem 2 (lower case indicates a mutated residue). C877 forms a Watson-Crick pair with g958 and C878 with g957. If protonated, C861 has a potential to form an additional base triple, C877-g958·C861+. Importantly, there are no interatomic clashes in this model.
Triple-helix models. Modeling was performed using miniCarlo as described in Materials and Methods. (A and B) Eleven triples are shown for poly(rU)-poly(rA) · poly(rU) (A) and poly(rC)-poly(rG) ·poly(rU) (B). In each strand, conformations of consecutive residues are identical. Watson-Crick strands are colored in brown (purine strand) and yellow (pyrimidine strand), and the third poly(rU) strand is colored in cyan. Backbones are schematically shown as ribbons, and sugar O4′ atoms are colored in red. (C) A typical U-A·U triple from the WT pseudoknot model. (D) A C-G·U base triple from the model of the pseudoknot structure for the S2.3+3′ mutant. (E) An example of a single C-G·U base triple found in the crystal structure of the Haloarcola marismortui 50S ribosomal subunit (1). (F) An example of a C-G·C+ base triple from a DNA triplex (PDB 149D) (23).
The model for the pseudoknot structure of the WT K. lactis sequence (Fig. 5A) was calculated in a similar way. Stem 2 is divided into two segments: the lower part consists of five U-A·U base triples, U876-A958·U862, U875-A960·U863, U874-A961·U864, U873-A962·U865, and U872-A963·U866, and the upper part consists of five base pairs, including four Watson-Crick pairs and the wobble G868-U967 pair (Fig. 5B). Residues C861, C877, and C957 are unpaired in this model. They are located inside stem 2 between the last triple (U876-A958·U862) and the terminal Watson-Crick pair (C878-G956), but their precise conformation cannot be predicted with certainty and may be quite flexible. Interestingly, these three unpaired cytosines are not conserved in the Kluyveromyces sequences (see Fig. 8). Residue U959 is bulged out between two base triples in the model. It is conserved in four out of six Kluyveromyces sequences.
A three-dimensional model for the wild-type pseudoknot structure. Modeling was performed using miniCarlo as described in Materials and Methods. (A) A ribbon representation (three different views). Stem 1 is shown in blue, Watson-Crick strands of stem 2 are colored in brown (purine-rich strand) and yellow (pyrimidine-rich strand), and the third oligo(U) strand is colored in cyan. Bulged-out U959 is shown in magenta; unpaired residues C861, C877, and C957 underneath the last triple are in red; and the Watson-Crick pair C878-G956 terminating stem 2 are in gray. (B) A schematic representation of base-pairing in the pseudoknot. Vertical lines represent Watson-Crick interactions; tilted lines, Hoogsteen interactions; and the asterisk, a G·U wobble pair. Note that if protonated, C861 has a potential to form a base triple (C878-G956·C861+).
Additional modeling calculations showed that a slightly different pseudoknot conformation with a comparable energy is also possible for the WT sequence. In this conformation, the register of the third strand is shifted by one position relative to the duplex, forming only four U-A·U triples and affecting mostly the conformation of the junction between the two stems (not shown).
One more modeling experiment was carried out to explain the moderate effect of the double transition mutation S2.3+3′ on dimer formation in vitro and on telomerase function in vivo, compared to the severe effects of the double-transversion mutations S2.2+2′ and S2.4+4′ (Fig. 2; Table 2) (32). Formation of a structure similar to that of the WT by the S2.3+3′ mutant would require the formation of C-G·U triples in the triple-helical part of stem 2. To the best of our knowledge, such triplexes have not yet been described in the literature. Surprisingly, computer modeling showed that a perfect triple helix can be formed by C-G·U triples, almost identical in conformation to the U-A·U triple helix (Fig. 4B). The H3 atom of the uracil from the third strand forms a single hydrogen bond with the guanine N7, and there is an additional weak hydrogen bond between the uridine hydroxyl group and the phosphate oxygen of the guanine (Fig. 4D). The electronegative atom O4 of the uracil is juxtaposed against the electronegative atom O6 of the guanine with a relatively short distance of 2.9 Å, resulting in a slight increase in the conformational energy. However, this increase was compensated for by other interactions in the triple helix. We found a similar single base triple (Fig. 4E) in the crystal structure of the Haloarcola marismortui 50S ribosomal subunit (1). In the ribosomal structure, this triple is isolated and unconstrained by the overall geometry of a triple helix. Thus, O4 and O6 are positioned at a more optimal distance of 4.0 Å. The high similarity between the conformations of the WT U-A·U and the mutant C-G·U triple helices is consistent with the relatively moderate effect of the corresponding mutation (S2.3+3′) on dimer formation (Fig. 3) and telomerase function (Fig. 2).
In summary, the triple-helix structure is consistent with several observations. First, double-transversion mutations in the triple-helical part of stem 2 (S2.2+2′ and S2.4+4′) failed to restore dimer formation in vitro (Fig. 3 and data not shown) and telomerase function in vivo (Fig. 2) (32). In contrast, double compensatory mutations in the double-helical part fully restored dimer formation (Fig. 3, S2.1+1′) and telomerase function (Fig. 1, S2.6+6′, and 1/1′ in reference 32). Second, single mutations in the CS3 loop completely abolished the dimer formation (Fig. 3, L.c) and telomerase function (Fig. 2, L.b, L.c, and L.d). The double-transversion mutations and the single mutations in the loop disrupted Hoogsteen hydrogen bonds and thus the triple-helix formation. Presumably, without the formation of the triple helix, the loop could not be accommodated anymore by the pseudoknot structure because it would clash with stem 2. Therefore, the interaction between CS3 and CS4 to form the pseudoknot structure was not possible in these mutants. Third, the transition double mutation S2.3+3′ had an intermediate effect, partially restoring the dimer formation (Fig. 3) and telomerase function (Fig. 2). Modeling studies revealed that this mutation could still retain a triple-helical structure composed of C-G·U base triples (Fig. 4B, D, and E), consistent with its intermediate effects on dimer formation and telomerase function.
Triple mutations.To test the triple-helix prediction, we combined three mutations, one in each strand of the triple helix (S2.3+3′+L.c and S2.0+0′+L.b) (Fig. 6A and B). In these mutants, the substituted cytosines in the third strand (L.b and L.c) must be protonated at their N3 positions to form C-G·C+ triples. Therefore, these base triples depend on low pH. Such triples (Fig. 4F) have been studied extensively for DNA (see, e.g., reference 23). For RNA, a single C-G·C+ triple was observed in stem 2 of the frameshifting pseudoknot from the beet western yellow virus (26). This base triple contributed to the pseudoknot stabilization in a pH-dependent manner (21). In our case, while mutation L.c alone completely abolished dimer formation (Fig. 3C), the combined mutation S2.3+3′+L.c fully restored it at pH 5 but not at pH 7 (Table 2; compare Fig. 6C to D). Not only were the amounts of dimers formed reduced at pH 7, but their electrophoretic mobility also was slightly increased, suggesting that their structure was altered. In contrast, the formation of dimers by the WT sequences was not pH dependent, neither in the amount of dimers formed nor in their electrophoretic mobility. When introduced into K. lactis cells, the loop mutations abolished telomerase function in vivo (Fig. 2, L.b and L.c). Strikingly, adding the corresponding double mutations S2.0+0′ or S2.3+3′ to the loop mutations L.b or L.c, respectively, partially restored telomerase function, as revealed by the normal colony phenotypes, the mildly shortened telomeres (60 to 80% of the WT length), and the incorporation of BclI telomeric repeats (Fig. 2 and Table 2). Furthermore, growing the yeast at low temperature (16°C) fully restored normal telomere length in both double and triple mutants but not in the loop (L.c) mutant (Fig. 2F). The ability to suppress the phenotype by low growth temperature suggested that the double and triple mutations reduced the pseudoknot stability rather than significantly altering its structure. When the 3-nt substitution in the CS3 loop was shifted by one nucleotide towards stem 1 (from L.c to L.b), the triple mutation still supported dimer formation in vitro, albeit with somewhat reduced stability (Fig. 6C and D and Table 2, S2.3+3′+L.b). This mutation was expected to fix the third strand in a different register relative to the duplex, consistent with the alternative conformation of the pseudoknot (see above). A small but reproducible change in the dimer mobility was observed for this mutation (Fig. 6C), suggesting that the global structure of the pseudoknot was altered. Strikingly, when introduced into K. lactis cells, S2.3+3′+L.b and a similar mutation, S2.0+0′+L.a, abolished or severely impaired telomerase function (Fig. 2). We observed a 43% reduction in the level of TER in the S2.3+3′+L.b mutant (Table 2), which could have reduced telomerase activity. However, we observed a reduction of 90% in the level of TER in an unrelated mutant in another region, while it was still active and able to maintain stable telomeres, even if very short (not shown). In addition, the reduction of 40% in the level of TER in another unrelated mutant caused only slight shortening of telomeres (not shown). Therefore, although the reduction in the level of TER may exacerbate the phenotype, it is clearly not sufficient to account for the complete telomerase deficiency of the S2.3+3′+L.b mutant.
pH-dependent dimer formation by oligoribonucleotides. (A and B) A schematic model of the K. lactis pseudoknot with five base triples (A), with the positions of the mutations (B) highlighted in gray. (C and D) Radioactively labeled CS4 and unlabeled CS3 (0.1 and 0.2 pmol, respectively) were analyzed as described in the legend to Fig. 3 and Materials and Methods. Identical gels were run simultaneously at pH 5 (C) and pH 7 (D). Single oligoribonucleotides are indicated by CS4 above and asterisks below the lanes. Lanes with mixtures of both CS3 and CS4 are indicated by the name (above) and number (below) of mutations introduced. (E) UV melting experiments were performed using various oligoribonucleotides at pH 5, 6, and 7. The Tm values were determined as described in Materials and Methods.
To study the sequences synthesized by the telomerase triple mutants, we cloned and sequenced at least 10 individual telomeres from each of the strains by using a ligation-mediated PCR method. Interestingly, we found truncated repeats in the S2.3+3′+L.c and S2.0+0′+L.a telomeres (one example for each mutant is shown in Fig. 7). Such truncated repeats were previously observed in the S2.3+3′ mutant, as well as in template boundary mutants (32), but never in any other telomere clones from wild-type or mutant strains. We did not find such truncated repeats in the S2.0+0′+L.b telomeres, but instead they contained mutations in the nascent portions of the telomere (Fig. 7). Although at present we do not have a mechanistic interpretation of these results, they nevertheless show that the structural details of the pseudoknot are crucial for telomerase function. The triple mutations not only reduced telomerase activity and telomere length but also altered the nature of telomere synthesis, indicating that the pseudoknot is intimately involved in telomerase action.
Incorporation of truncated and mutated repeats onto telomeres in vivo. Shown are examples of cloned telomere sequences from the S2.3+3′+L.c, S2.0+0′+Lb, and S2.0+0′+La mutant strains at their sixth passage after introducing the mutant ter gene. The telomeric repeats are aligned to the template sequence above. Underlined are the repeated sequence at the beginning and the end of the template. Underlined in the S2.3+3′+L.c and S2.0+0′+La telomeres are nucleotides used for base-pairing and initiating the synthesis of truncated repeats from the middle of the template. The telomerase template and wild-type telomeric repeats are shown in black; BclI repeats synthesized by the mutant telomerase, in blue; truncated repeats and misincorporated nucleotides, in red. The BclI mutations are highlighted in green.
We tested additional combinations of double mutations, and all of them abolished telomerase function, further confirming that the pseudoknot structure is essential (Fig. 2 and Table 1, S2.3′+L.c, S2.3+L.c, S2.0′+L.b, and S2.0′+L.a). Altogether, in vitro and in vivo analyses of various disruptive mutations and compensatory mutations forming C-G·U or C-G·C+ base triples confirmed the triple-helix model and indicated that not only the stability but also the precise structure of the pseudoknot is essential for telomerase function.
Thermal melting.We next monitored the thermal denaturation of oligoribonucleotides in solution by following the absorption at 260 nm along a temperature gradient. A cooperative transition was observed for the mixture of WT CS3 and CS4 sequences, presumably indicating the melting of the CS3-CS4 dimers. No such a transition was observed for each sequence alone (not shown). The Tm of the WT dimer was only slightly affected by pH (50 to 55°C at pH 7 to 5) (Fig. 6E and Table 2). Similar pH-independent Tm values of 55°C and 56 to 57°C were observed for the double mutants S2.0+0′ and S2.3+3′, respectively, yet these mutations only partially restored dimer formation as observed by native PAGE (Fig. 6D and E) and telomerase function in vivo (Fig. 2C and D). This suggested that although they have similar midpoints of dissociation (Tm values), mutant constructs might be less stable than the WT at room temperature. Mechanical forces may enhance the dissociation of the mutant dimers during prolonged gel electrophoresis under such circumstances, as has been observed for kissing hairpin complexes of the human immunodeficiency virus type 1 dimerization initiation site (27). Adding the third mutation significantly increased the stability of the structure in a pH-dependent manner (Fig. 6 and Table 2). At pH 7, the triple mutations S2.0+0′+L.b and S2.3+3′+L.c had Tm values similar to those of the double mutations (55°C). However, these values dramatically increased to 65°C and 71°C, respectively, at pH 5, consistent with the protonation of cytosines and the formation of C-G·C+ triples. This was consistent with the efficient dimer formation of the triple mutants at pH 5, as observed by native PAGE. However, these Tm values were significantly higher than that of the WT. In addition, the corresponding triple mutants only partially restored telomerase function in vivo (Fig. 2). One can think of several possible explanations. If the intranuclear pH was higher than the pKa of the substituted cytosines in the CS3 loop, these cytosines would not be protonated and would not form triples. Indeed, at pH 7 these mutants had Tms similar to those of the double C-G·U mutants and behaved similarly on native PAGE, except for the increased mobility of the C-G·C mutants at pH 7. They also had similar telomerase activity in vivo. If, on the other hand, the intranuclear pH is low enough to allow protonation of the C residues, the inability to fully restore telomerase function may indicate the overstabilization of the structure, supporting the conformational switch model (6). However, the suppression of the telomere phenotype by growing the triple mutant at low temperature (16°C) argues against this possibility. Finally, the internal pH may be close enough to the pKa values to allow partial protonation and partial formation of the correct structure.
Conservation of the triple-helix structure between K. lactis and human.During the course of our research, an independent work has been published, reporting on the nuclear magnetic resonance solution structure of the human telomerase pseudoknot (29). Remarkably, this structure revealed features similar to the 3D model of the K. lactis pseudoknot reported here, most importantly, the presence of a triple helix within stem 2. At the same time, there are many significant differences between the two structures. There are five consecutive U-A·U triples in the K. lactis pseudoknot and only three in the human pseudoknot. Stem 2 is longer (12 bp) and the loop is shorter (6 nt) in K. lactis than in human (9 bp and 8 nt, respectively). Therefore, the loop in the K. lactis pseudoknot is much more constrained and structured. The details of the junction between stems 1 and 2 are also quite different. In K. lactis, stem 2 ends with a Watson-Crick G·C pair, there is a triple of unpaired cytosines just next to it, and the helical twist angle between stems 1 and 2 is 56°. In human, there is a non-Watson-Crick A·U pair at the junction of the two stems, and the twist angle is 75 to 80°. The human structure was solved for a mutant RNA construct in which a bulged nucleotide in stem 2 (U177) was deleted. Its deletion was suggested previously to reduce human telomerase activity (to 10% of the WT activity) by stabilizing the pseudoknot structure and impairing the function of the pseudoknot as a molecular switch (6). However, the reduction in telomerase activity of the U177 deletion mutant may also result from an alteration of the pseudoknot structure, rather than from the increased pseudoknot stability. As described above for the K. lactis pseudoknot, one nucleotide shift in the register of the triple helix, presumably affecting the junction between stem 1 and 2, completely abolished telomerase function (Fig. 2, compare S2.3+3′+L.c to S2.3+3′+L.b and S2.0+0′+L.b to S2.0+0′+L.a). These results demonstrated how crucial are at least some aspects of the pseudoknot structure. In K. lactis, the length of stem 2 (12 bp) and the increased stability due to the triple-helix formation argue against the involvement of stem 2 in a conformational switch. However, a shift between different registers of the triple helix is theoretically possible. If such a switch was required, fixing the structure in either conformation would impair telomerase function. Such a model can explain why the triple mutations that preserved a stable triple helix in vitro failed to fully restore telomerase function in vivo. However, without experimental evidence this remains a speculation.
Conclusion.We carried out a detailed analysis of the pseudoknot structure in K. lactis telomerase RNA. The successful integration of phylogenetic, molecular, and genetic analyses with molecular modeling yielded a 3D structure, which in turn was confirmed by additional mutational analyses in vitro and in vivo. Although the calculated model is not a high-resolution structure, its accuracy, predictive power, and in vivo relevance were demonstrated. Computer modeling is not routinely used to predict 3D structures of complex RNA molecules. Nevertheless, its usefulness is clear. One example is a computer prediction of the 3D architecture of group I catalytic introns (19), two decades before their crystal structures were solved (reviewed in reference 35). The success in developing a 3D model for the K. lactis pseudoknot was largely due to the highly constrained nature of this structural element: only 4 out of 41 residues are not part of base pairs or triples (Fig. 5B). This model is also supported by the conservation found in the pseudoknot within TER sequences of the "Kluyveromyces marxianus" cluster of species (Fig. 8). Furthermore, it is consistent with experiments with S. cerevisiae (3, 10, 13) suggesting that a similar U-A·U triple helix also forms in TLC1. While the homology of stem 2, including the triple helix, is apparent for K. lactis, S. cerevisiae, and human sequences, there are possible alternative arrangements for stem 1 in S. cerevisiae. In addition, while stem 2 and the junction between stems 1 and 2 are critical for telomerase function, mutations in the predicted stem 1 seem to be less detrimental (Fig. 1). Deletions in the sequences corresponding to stem 1 in S. cerevisiae had little or no effect on telomerase function (3, 13). Altogether, these observations suggested that stem 2 with the triple helix is the most crucial and conserved part of this element. In some species it may be sufficient to provide the essential function of this domain. Strikingly, the much smaller ciliate pseudoknot can partially substitute for the S. cerevisiae and human domains (3, 16), suggesting the presence of this conserved functional element in all telomerases.
Conservation of the triple-helix pseudoknot in the "Kluyveromyces marxianus" cluster of species. Pseudoknot models are shown for the TER sequences of six Kluyveromyces species (32). Conserved residues are highlighted in gray. Hoogsteen interactions are indicated as tilted lines.
A major-groove triple helix was proposed in 1957 for the synthetic polymer poly(rU)-poly(rA)·poly(rU) (8). Those authors suggested that "this three-stranded structure may have significance as a prototype for a biologically important three-stranded complex." Isolated base triples have been found in RNA structures. However, to the best of our knowledge, the telomerase RNA pseudoknot is the first example found in a native RNA molecule of consecutive U-A·U triples forming a major-groove triple helix. Furthermore, it represents the first structural element conserved between the otherwise highly divergent yeast and human telomerase RNAs.
In the telomerase catalytic core model, the pseudoknot is juxtaposed to the telomerase template by the core-enclosing helix (13). Interestingly, cloned telomeres from the triple-helix mutants revealed defects in processivity and fidelity of the enzyme (Fig. 7) (32). These aberrations in telomere synthesis suggested that the pseudoknot is intimately involved in the telomerase reaction. Experiments with S. cerevisiae indicated that pseudoknot sequences are important for Est2 binding (3, 13, 15), but no indication for a direct interaction with the pseudoknot sequences was found. Since this unusual triple-helical pseudoknot is conserved between human, yeast, and possibly also ciliate telomerases but has not yet been found elsewhere in nature, we propose that it contributes an essential function, conserved and unique to the telomerase ribonucleoprotein, which is yet to be uncovered.
This work was supported by United States-Israel Binational Science Foundation grant 2001065 to Y.T. and N.B.U. and by Israel Science Foundation Grant 676/02 to Y.T.
We thank Tom Goddard, Tom James, Elly Ordan, Debbie Shalev, Joseph Shlomai, Ruth Sperling, and Eric Westhof for critical reading of the manuscript and for stimulating conversations. N.B.U. is indebted to Tom James for his encouragement and continuing support.
Received 26 September 2006.
Returned for modification 9 November 2006.
Accepted 19 December 2006.
↵▿ Published ahead of print on 8 January 2007.
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Molecular and Cellular Biology Feb 2007, 27 (6) 2130-2143; DOI: 10.1128/MCB.01826-06
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