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Ribonucleic acid is the middleman in the process whereby deoxyribonucleic acid, the primary genetic material, is translated into protein, the structural and functional material of all life. As is appropriate to a middleman, the study of RNA was all but neglected until the last decade. 'Tor years we've been thinking of RNA as nothing but an information tape or a way of stringing proteins together/ says Norman Pace of the National Jewish Hospital in Denver. "Now we're finding it's a much more powerful molecule than that/' In both DNA and RNA the sequence of nucleotides, or bases, which are the letters of the genetic alphabet, encodes the recipe for protein. Each protein is encoded in a stretch of bases that makes up a gene. In DNA, other base sequences appear in a region located upstream from each gene or group of genes. These sequences are the signals that regulate the production of protein by controlling the transcription of DNA Into RNA. Scientists also are finding that RNA often can provide its own instructions for the synthesis of protein, that is when to make protein and how much to make. Rather than the arrangement of bases, however, It Is the structure of the RNA, the way it folds Itself into helices, that embodies much information directing a cell's functioning. It is as if kinks appeared in the tape to start a tape recorder when it was time for the music to play and again when it was time to stop. The difference between RNA and DNA that makes structure important in RNA and nucleotide sequence important in DNA is that RNA is single-stranded and DNA is double-stranded. The two strands of the DNA double helix connect at every link In the chain of nucleotides. The result is a stiff and unreactive molecule, one that must communicate principally through base-sequence codes. By contrast, because it is single-stranded, RNA can use form to function as well. Singlestranded RNA folds upon Itself, becom- 34 MOSAIC by David Holzman Unlike its twin-stranded cousin, RNA uses structure to help it perforin its function. ing double-stranded In places and forming three-dimensional structures called helices that are able to convey their own messages in the cell. Base pairing produces the helices in RNA. In the DNA base pairing, adenine (A) binds to thymine (T), and guanine (G) binds to cytosine (c). [In RNA, uracil (u) replaces thymine.] The twin-stranded DNA exhibits full complementarity; every base in each strand is paired. Helix formation occurs in RNA when a moderate amount of complementarity exists between two nearby pieces of the same strand, which twists on itself as complementary bases pair. These helices are called the secondary structure of RNA. They are represented as two-dimensional structures called hairpins. In addition RNA forms tertiary structures when single-stranded loops on the ends of hairpins pair with other loops on the strand, orienting the hairpins three dimensionally. just as rivers descend from the heights and lose potential energy as they drop, the pairing arrangements in RNA may shuffle spontaneously until the molecule lands in the structure with the least free energy, a thermodynamic sea level. TInoco measured the strengths of helices having different base sequences. This task was not easy, because the strength of any pair, say A with u, is influenced by what the adjacent bases are, and even by the neighbors beyond those. These pairing strengths can then be integrated into a computer program that can find the most stable pairing arrangement for an RNA molecule. Although Tinoco's rules can give a reasonable first approximation of an RNA structure, they do have several serious shortcomings. First, Investigators have not tested all possible short sequences, so the rules are based on considerable extrapolation. Second, the strength of the pairing is not the sole determinant of structure. The order in which an RNA chain forms affects structure, because bases form pairs as the RNA chain grows; they do not wait until the chain is fully formed. A particularly strong helix early in the chain could prevent the molecule from reshuffling its helices into a more stable structure when fully formed. A third shortcoming of Tinoco's rules is that they do not analyze tertiary structure. Computer attempts to impose a two-dimensional structure on a molecule, neglecting three-dimensional pairing, frequently yields false results. Tinoco's rules Thermodynamic analyses of this kind, based on comparison of the strengths of the bonds between molecules, are applied quite successfully to secondary structure where they can explain many observations of the regulation of genetic functioning by RNA. This is because the Ignacio TInoco of the University of California at Berkeley has devised a set of rules for predicting the structural folding of an RNA from the sequence of its bases. Tinoco's rules are based on the idea that Stop sign. Stem and loop at the end of an RNA transcript is thought to signal transcription termination. Asterisks indicate sites of norma! mutations, which can disrupt the structure and eliminate termination. C~G C-G A~U GUUCAG regulatory end of an RNA strand appears to be too short to fold on itself, and so it retains its secondary structure. It is also because ribosomes translating the RNA genes can prevent tertiary interaction with the rest of the chain, maintaining a tractable two-dimensionality. Although some biochemists speak condescendingly of computer studies of RNA folding, it is possible to piece together quite accurate secondary structures by supplementing computer studies with biochemical studies. If linoco's rules say a helix occurs at a certain point in the RNA, an enzymatic probe that cuts RNA only at double-stranded sites will show whether the helix is really there. Genetic analysis, testing the effect of a mutation in paired bases on the strength of the helix they help to form, can provide the same information, but with less certainty. Stop signs In messenger RNA a region called the leader controls gene expression. Once the enzymes, called polymerases, transcribe the RNA leader from DNA, the leader folds itself into shapes that the enzymes recognize as signals. The most basic signal is a small hairpin with a loop at Its end. The enzyme, recognizing the loop structure as a stop sign, pauses briefly before being directed further In the transcription process. If the products of the genes are not needed, the process ends and the RNA separates from Its template. If they are needed, transcription continues Into adjacent genes. 36 MOSAIC Researchers had started to come upon these stop signs, called terminators, in the mid-1960s. That was long before anyone suspected that RNA structure had anything to do with the mechanics of terminators. Charles Yanofsky of Stanford University had a typical experience. When his student Ethel Jackson deleted genetic material in the then-mysterious region between the promoter, which binds the enzyme to start transcription, and the genes, it was as if she had pulled a plug. Suddenly the group of genes following the promoter was making five times as much protein as predicted by what were then current theories of gene regulation. Nevertheless terminators attracted little notice for some years. In the early to middle 1970s, recalls Martin Rosenberg, now of Smith Kline and French Research Laboratories in Philadelphia, "everyone thought that most regulation occurred at the promoter. There were about 100 scientists studying promoters, and Charlie Yanofsky and I were studying terminators. Everyone thought terminators were just going to be the period at the end of the sentence/' Rosenberg made the first definitive connection between the structure and function of terminators in 1974. He had been studying termination in p h a g e Lambda, a favorite laboratory virus. In the phage one terminator controls a set of genes that is necessary for a stage in the phage's life cycle. Only a small percentage of phages enter this stage, and the terminator helps control the relative numbers by having what can be considered a slow leak. Rosenberg had two sets of mutant phages. One Increased termination, leading to fewer phages entering this second stage of development, and one reduced it, leading to more phages entering. Using newly developed techniques, Rosenberg determined the sequence of nucleotides in the terminator, hoping to find clues to its operation. He discovered two strikingly complementary sets of bases in the terminator, implying that a helix might form. More importantly, he discovered that the termination-enhancing mutant changed a genetic letter to strengthen the helix, while the leaky mutants weakened it. Over the next few years the evidence grew stronger that hairpins cause termination. Biochemical experiments proved beyond a doubt that the structures formed in terminators. Terry Piatt, at Yale University, inserted synthetic hairpins into the middle of a gene and found that the stronger the hairpin, the greater the frequency of termination. Pausing These experiments demonstrated that hairpins are involved in termination, but not how they are involved. Two experiments in the middle to late 1970s led to the current model of how termination takes place. There are two kinds of terminators, one requiring a protein to catalyze the separation of nascent RNA from the DNA template, and the other having no such requirement; this protein,, discovered by Jeff Roberts, was called rho. In one experiment, Rosenberg found that in the absence of rho, a polymerase would pause at a rho-dependent terminator for 30 to 40 seconds. Then It would continue making RNA. In the second, Berkeley's Mike Chamberlin showed that this pausing is not restricted to termination sites, but occurs wherever messenger RNA can form helices. This discovery led to the idea that hairpins cause pausing. Terry Piatt describes the generally accepted speculation about this process: Recent evidence strongly suggests that at any one time about 12 nucleotides of a growing RNA chain are always paired with the DNA template inside the RNA polymerase. Then, says Piatt, hairpin formation rips the RNA from its DNA template. The resulting helix jams the enzyme. The pause is thought to provide the time necessary for the RNA chain-release reaction to take place. In the rho-dependent terminators, this amounts to giving rho enough time to interact with the polymerase complex and to catalyze chain separation. In the rho-independent terminators, the m e c h a n i s m is more obvious. These terminator hairpins are followed Immediately by a string of u bases, which, as determined by Francis H. Martin, now of Applied Molecular Genetics Incorporated, and Ignacio TInoco In 1981, provide an extraordinarily feeble link to the As of DNA template. In r h o - i n d e p e n d e n t terminators, therefore, the pause is thought to halt transcription once the string of us has been transcribed and while it is still paired Inside the enzyme with Its DNA template. The A-u bonds rupture, causing the RNA to fall away from the DNA. W h e n expression of d o w n s t r e a m genes Is needed, transcription continues past the terminator. In some cases this can h a p p e n because antitermination proteins override the terminator. In other cases, where terminators function like leaky plugs to moderate the number of downstream genes to be expressed, terminators probably fail to cause pausing. This may happen because either the terminators fail to fold during transcription or they fold then spontaneously unfold. Either case would be consistent with evidence that frequency of termination is related to the strength of the helix. Attenuation In a special class of terminators called attenuators, the RNA takes a much more MOSAIC 37 active role in its own transcription. The heart of the attenuator is still the basic terminator, b u t instead of being acted upon by antitermination proteins, the terminator competes with an intrinsic antiterminator for use of some of the nucleotides letters of its helix. When the bacterium n e e d s the products of the downstream genes—which in one thoroughly studied case are the enzymes that make the amino acid tryptophan—the antiterminator helix comes together. This action masks some of the nucleotides that would normally fold into the terminator helix and permits transcription to proceed. Two types of attenuator have been found. One regulates protein synthesis by controlling transcription, and the other regulates by controlling translation. The mechanics of the process are intricate: In each case individual segments of RNA fold to produce either a terminator or an antiterminator, but never both at the same time. Which pattern occurs depends in part on the circumstances of the cellular environment. One example of transcriptional attenuation involves a gene that makes the enzymes that synthesize the amino acid tryptophan. In this case the production of the amino acid is tied to the level of tryptophan in the cell. High levels of the amino acid favor the formation of a terminator, and low levels favor the formation of an antiterminator. When an RNA strand assumes the terminator shape, the polymerase falls off the leader instead of transcribing the downstream genes necessary for continued production of tryptophan. When tryptophan levels fall a n d the m e s s e n g e r RNA assumes the antiterminator shape, then downstream genes are free to be transcribed and tryptophan production can increase once again. Even when tryptophan is a b u n d a n t , however, these genes are not always blocked; tryptophan production will never shut down completely. An example of translational attenuation occurs in bacteria resistant to the antibiotic erythromycin, a drug that poisons ribosomes. In the absence of the antibiotic, a terminator forms in these bacteria, blocking translation of RNA to a protein that makes ribosomes immune to erythromycin. As is the case with trypt o p h a n , however, p r o d u c t i o n never Holzman is a Washington-based science writer who works most frequently in biology. 38 MOSAIC message by surviving ribosomes may begin. These survivors then translate immunity protein in bulk, producing more immune ribosomes and quickly building up.the bacterium's resistance. Kinetic analysis shuts down completely. Some immunity-producing protein is always incorporated into a few ribosomes, making them immune to erythromycin. The few immune ribosomes produced in the absence of erythromycin become important later when the drug, floods the cell. Then messenger RNA assumes its antiterminator shape, and translation of the Despite the success scientists have had in explaining attenuation and termination with t w o - d i m e n s i o n a l t h e r modynamic analysis, some recent observations in messenger RNA defy analysis. Most scientists studying RNA now think that a complete u n d e r s t a n d i n g of Its properties will require a better knowledge of the relative importance of kinetics and t h e r m o d y n a m i c s In RNA structure formation, a better knowledge of which proteins interact with messenger RNA and how they Interact, and an understanding of the three-dimensional, tertiary structure of RNA. The rate, or kinetics, of the chemical and biochemical reactions may influence the outcome of regulation by messenger RNA as much as or more than the predictions that could be made by applying Ignacio Tinoco's thermodynamic rules. Tinoco's rules predict the structure an RNA would assume if it had all the time in the world to descend to thermodynamic sea level. Regulatory events such as termination and attenuation, though, take place in fractions of a second on chains that are growing at 50 or 100 nucleotides per second and folding into helices three orders of magnitude more quickly. "We don't know exactly what structures are being formed as the RNA is made," says Berkeley's Mike Chamberlin. The data certainly s u g g e s t , however, that thermodynamics is not the whole story. Yale's Terry Piatt had been puzzled by the observation that a single nucleotide mutation In the stem of a hairpin that changed the strength of the hairpin only slightly could drastically reduce the frequency of termination at that site. "I didn't understand this until I realized that hairpin formation is almost certainly a kinetic phenomenon," he says. "If you introduce a single base-pair mismatch, It's like missing a tooth in a zipper." Chamberlin thinks that is putting the case too strongly. He has watched many RNA chains grow In vitro, In cell-free, artificial systems, in his attempts to correlate the strength of hairpins with the percentage of RNAs that pause at each and the duration of the pauses. "None of the hairpins we see implicated in pauses are perfect hairpins," Chamberlin says. "If Piatt were right, we wouldn't expect any of those to cause pausing. I think the effect could be kinetic but there's obviously a thermodynamic component." In recentf studies Chamberlin, as well as Don Mills and Susan LaFlamme at Columbia University's College of Physicians and Surgeons, have found strong hairpins without associated p a u s e s . These findings have led to more quandaries about what structures are significant in a dynamic context. Chamberlin's data come from his further studies of growing messenger RNA chains, while Mills and LaFlamme's data come from experiments that LaFlamme did while preparing her Ph.D. dissertation. The Columbia researchers used recombinant DNA techniques to make a DNA template for a small RNA that is normally copied from its own RNA template. The molecule is not a messenger RNA, which means that its structure is unencumbered by ribosomes translating protein. The entire chain is free to bunch and tangle. In the experiments the growing chain paused at only one of the molecule's many strong hairpins. Hairpins, Chamberlin p o i n t s out, "may not form rapidly enough to be sig- clear magnetic resonance will ultimately determine what structures are present in the growing RNA chains. This determination will make it possible to ascertain the relative contributions of kinetics and thermodynamics. Cause for pause nificant in terms of the elongating complex." His observation is particularly salient because without the ribosomes on the molecule, stable structures could be preventing new helices from forming quickly at the growing end of the RNA. Chamberlin says that studies with nu- These future studies should clear up the mystery of hairpins without pauses, but scientists are unsure about where to look for solutions to the mystery of pauses w i t h o u t h a i r p i n s . Mills and LaFlamme have found two consecutive pause sites downstream from a hairpin, although the sites were not related to that or any other hairpin. They tested this finding by removing the hairpin, inserting in its place 51 bases lacking two-dimensional structure, and removing most of the molecule downstream from the pauses. During these manipulations they left intact only a small number of bases on either side of the pauses. The pauses persisted throughout these manipulations. The Columbia researchers believe that local factors within a few bases of the pause sites were responsible. The most specific explanation Mills can offer, though, is the possibility that Protein-binding sites in RNA Some proteins have jobs in the cell that involve binding DNA or RNA. Many of these proteins regulate their own number in a unique way. When they become so numerous in the cell that they have saturated their natural binding sites, they begin to bind to their own messenger RNA in a way that prevents their own synthesis by ribosomes. This binding happens because, in their messenger RNA, repression sites have apparently evolved to imitate the structure of normal cellular binding sites. This molecular mimicry is imperfect, though, so the proteins do not begin binding their messages until ail their normal binding sites are saturated. The proteins appear to find their RNA binding sites by recognizing the secondary structure but not the order of bases in helical regions. In experiments on a repressor site in the messenger RNA of phage R17, Olke Uhlenbeck of the University of Illinois changed just about every nucleotide in turn to find how proteins recognize their binding sites. When he changed any base in a way that destroyed pairing, the protein failed to bind. Changing two bases at a time in a way that preserved the pairing, however, allowed the protein to bind to the site with normal strength. Only four of the single-stranded residues, or segments, were necessary for binding, explains Uhlenbeck, so "the protein recognizes the hairpin by looking at the singlestranded residues held in a precise orientation by the helix." He was able to test this model by making a molecule with a very different sequence but the same structure. He showed that it still b o u n d protein, and he called it "wierdmer." This finding is consistent with the structural analysis of nucleic-acid helix shape performed by Norman Pace of the National Jewish Hospital in Denver. In the RNA helix, Pace explains, the chemically unique properties of the bases are sequestered well beneath the backbone of the molecule. If proteins recognize RNA secondary structure, Larry Gold of the University of Colorado thinks they can also recognize its conspicuous absence. Gold has studied regulation of a protein whose job in the phage T4 is to bind to single-stranded DNA during the phage's DNA-copying process, presumably to protect it from DNA-cutting enzymes. When the growing DNA becomes saturated, excess protein binds to its own message at a site that, like the DNA, is conspicuous for its lack of structure. Gold believes the protein interacts with the nucleic-acid backbone, which is quite similar in DNA and RNA. "The reason the protein can see the backbone is that there is no secondary structure to interfere," says Gold. Peter Model of Rockefeller University claims that the protein recognizes sequences of nucleotides in DNA and RNA, not the absence of structure. Gold does not think Model is right, but he plans to make sure. He is constructing a wierdmer for this protein just as structureless as the real thing but with an entirely different sequence. • MOSAIC 39 the particular sequence of bases "could result in some kind of stacking situation/' Chamberlin, responding to pauses without hairpins that turned up in his own messenger RNA studies as well as in the Columbia experiments, thinks that "the fairest thing to say is we know hairpins can cause pausing, but whether other structures can also do that or not is up in the air. We have no idea what the other structures would be. At this point we're not even sure there are other structures, but we can't rule them out." To complicate matters there are proteins and other molecules in living cells that are known both to enhance pausing and to depress it. Chamberlin had noted early in his pausing experiments that RNA grew much more slowly in artificial, cellfree systems than in living cells, and that the difference was entirely attributable to longer pausing. Postulating a pause suppressor in the living cell, he added a small protein called nusA to. his templating broth. This action resulted in still longer pausing. Adding an extract from the cell shortened the pauses. The suppressor obviously appears to be there, but Chamberlin still has not identified it. He speculates that other extraneous cellular molecules may be responsible for pause sites dissociated from hairpins, perhaps by altering the way the transcribing enzyme sees structure. Polarity sites As is the case for hairpinless pause sites, certain termination sites that require catalysis by rho also lack associated hairpins. This special class of rho-dependent sites plays a unique regulatory role in the cell. Such sites halt transcription of RNA just as ordinary terminators do, but in the middle of genes and only when the just-transcribed RNA is defective. The mechanism for termination at these sites, which are called polarity sites, depends on the fact that proteinmaking ribosomes in bacterial cells follow closely b e h i n d the polymerase, translating protein from the newly formed RNA. If a ribosome encounters a nonsense codon, it falls off the RNA. Transcription then is terminated at the next available polarity site. Scientists suspect that termination can h a p p e n only after the ribosomes drop off and their presence no longer blocks access of the rho protein to its target terminator. Though all this in part explains termination at polarity sites, it fails to address pausing there. Some researchers think 40 MOSAIC the cause of pausing at polarity sites will not become clear until they can learn to determine three-dimensional structure. The problem is that messenger RNAs are too scarce and too large to be subjected to conventional m e t h o d s for analyzing three-dimensional structure. The notion that three-dimensional structure plays some role in polarity is attractive, says Martin Rosenberg, partly because the absence of ribosomes between the mutation and the polarity site would leave a relatively long stretch of messenger RNA free to take on three-dimensional structure. "As soon as you stop translating," he says, "you have an RNA that will assume some stable tertiary structure. The formation of that structure may have a lot to do with inducing polarity," he says. Rosenberg adds that, compared to the mechanism of attenuation, which is unique to certain operons, or groups of genes regulated in common, "polarity is a more global mechanism. It operates within many of the translated operons in an entire cell. It's probably far more subtle, since it has to take into account many different translated operons." But, he adds, "we do not yet really understand how polarity works." "Tertiary structure is a way we get around explaining w h y all the pause sites don't have secondary structure," he says. • The National Science Foundation contributes to the support of research discussed in this article through its Biochemistry and Genetics Programs. * U. S. GOVERNMENT P R I N T I N G O F F I C E : 1984 461-633/20000