An early RNA world (Chapter 13) PDF

# An Early RNA World Life requires two basic functions. First, living organisms must be able to store and faithfully transmit genetic information during reproduction. Second, they must have the ability to catalyze the chemical transformations that drive life processes. A long-held belief was that t...

# An Early RNA World Life requires two basic functions. First, living organisms must be able to store and faithfully transmit genetic information during reproduction. Second, they must have the ability to catalyze the chemical transformations that drive life processes. A long-held belief was that the functions of information storage and chemical transformation are handled by two entirely different types of molecules: genetic information is stored in nucleic acids, whereas chemical transformations are catalyzed by protein enzymes. This biochemical dichotomy created a dilemma: Which came first, proteins or nucleic acids? If nucleic acids carry the coding instructions for proteins, how could proteins be generated without them? Nucleic acids are unable to copy themselves, so how could they be generated without proteins? If DNA and proteins each require the other, how could life begin? This apparent paradox was answered in 1981 when Thomas Cech and his colleagues discovered that RNA can serve as a biological catalyst. They found that some RNA molecules from the protozoan *Tetrahymena thermophila* can excise 400 nucleotides from its RNA in the absence of any protein. Other examples of catalytic RNAs have now been discovered in different types of cells. Called *ribozymes*, these catalytic RNA molecules can cut out parts of their own sequences, connect some RNA molecules together, replicate others, and catalyze the formation of peptide bonds between amino acids. The discovery of ribozymes complements other evidence suggesting that the original genetic material was RNA. ## Self-replicating ribozymes Self-replicating ribozymes probably first arose between 3.5 billion and 4 billion years ago and may have begun the evolution of life on Earth. Early life was probably an RNA world, where RNA molecules served both as carriers of genetic information and as catalysts that drove the chemical reactions needed to sustain and perpetuate life. These catalytic RNAs may have acquired the ability to synthesize protein-based enzymes, which are more efficient catalysts. With enzymes taking over more and more of the catalytic functions, RNA probably became relegated to the role of information storage and transfer. DNA, with its chemical stability and faithful replication , eventually replaced RNA as the primary carrier of genetic information . Nevertheless , RNA is either produced by or plays a vital role in many biological processes , including transcription , replication , RNA processing , and translation. Research in the past 20 years has also determined that newly discovered small RNA molecules play a fundamental role in many basic biological processes, demonstrating that life today is still very much an RNA world. These small RNA molecules will be discussed in more detail in Chapter 14. ## The Structure of RNA RNA, like DNA, is a polymer of nucleotides, each consisting of a sugar, a phosphate group, and a nitrogenous base, joined together by phosphodiester bonds (see Chapter 10). However, there are several important differences in the structures of DNA and RNA. Whereas DNA nucleotides contain deoxyribose sugars, RNA nucleotides have ribose sugars (Figure 13.1a). With a free hydroxyl group on the 2-carbon atom of the ribose sugar, RNA is degraded rapidly under alkaline conditions. The deoxyribose sugar of DNA lacks this free hydroxyl group, so DNA is a more stable molecule. Another important difference is that the pyrimidine uracil is present in RNA instead of thymine, one of the two pyrimidines found in DNA. A final difference in the structures of DNA and RNA is that RNA usually consists of a single polynucleotide strand, whereas DNA normally consists of two polynucleotide strands joined by hydrogen bonding between complementary bases (although some viruses contain double-stranded RNA genomes, as discussed in Chapter 9). Although RNA is usually single stranded, short complementary regions within a nucleotide strand can pair and form secondary structures (Figure 13.1b). These RNA secondary structures are often called hairpins (or hairpin-loop or stem-loop structures). When two regions within a single RNA molecule pair up, the strands in those regions must be antiparallel and with pairing between cytosine and guanine and between adenine and uracil (although occasionally guanine pairs with uracil). The formation of secondary structures plays an important role in RNA function. Secondary structure is determined by the base sequence of the nucleotide strand, so different RNA molecules can assume different structures. Because their structure determines their function, RNA molecules have the potential for tremendous variation in function. With its two complementary strands forming a helix, DNA is much more restricted in the range of secondary structures that it can assume and so has fewer functional roles in the cell. Similarities and differences in DNA and RNA structures are summarized in Table 13.1. ## Classes of RNA RNA molecules perform a variety of functions in the cell. Ribosomal RNA (rRNA) and ribosomal protein subunits make up the ribosome, the site of protein assembly. We’ll take a more detailed look at the ribosome in Chapter 14. Messenger RNA (mRNA) carries the coding instructions for a polypeptide chain from DNA to a ribosome. After attaching to the ribosome, an mRNA molecule specifies the sequence of the amino acids in a polypeptide chain and provides a template for the joining of those amino acids. Large precursor molecules, which are termed pre-messenger RNAs (pre-mRNAs), are the immediate products of transcription in eukaryotic cells. Pre-mRNAs are modified extensively before becoming mRNA and exiting the nucleus for translation into protein. Bacterial cells do not possess pre-mRNA; in these cells, transcription takes place concurrently with translation. Transfer RNA (tRNA) serves as the link between the coding sequence of nucleotides in an mRNA molecule and the amino acid sequence of a polypeptide chain. Each tRNA attaches to one particular type of amino acid and helps incorporate that amino acid into a polypeptide chain (as described in Chapter 15). Additional classes of RNA molecules are found in the nuclei of eukaryotic cells. Small nuclear RNAs (snRNAs) combine with small protein subunits to form small nuclear ribonucleoproteins (snRNPs, affectionately known as "snurps"). Some snRNAs participate in the processing of RNA, converting pre-mRNA into mRNA. Small nucleolar RNAs (snoRNAs) take part in the processing of rRNA. Two types of very small and abundant RNA molecules found in the cytoplasm of eukaryotic cells, termed microRNAs (miRNAs) and small interfering RNAs (siRNAs), carry out RNA interference (RNAi), a process in which these small RNA molecules help trigger the degradation of mRNA or inhibit its translation into protein. More will be said about RNA interference in Chapter 14. Another class of small RNA molecules are Piwi-interacting RNAs (piRNAs; named after Piwi proteins, with which they interact). Found in mammalian testes, these RNA molecules are similar to miRNAs and siRNAs; they have a role in suppressing the expression of transposable elements (see Chapter 18) in reproductive cells. Long noncoding RNAs (lncRNAs) are relatively long RNA molecules found in eukaryotes that do not code for proteins. They provide a variety of functions, including regulation of gene expression. In prokaryotes, an RNA interference-like system has been discovered, in which small CRISPR RNAs (crRNAs) assist in the destruction of foreign DNA molecules. Some of the different classes of RNA molecules are summarized in Table 13.2. ## Transcription is the synthesis of an RNA molecule from a DNA template All cellular RNAs are synthesized from DNA templates through the process of transcription (Figure 13.2). Transcription is in many ways similar to the process of replication, but a fundamental difference relates to the length of the template used. In replication, all the nucleotides in the DNA molecule are copied, but in transcription, only parts of the DNA molecule are transcribed into RNA. Because not all gene products are needed at the same time or in the same cell, the constant transcription of all of a cell’s genes would be highly inefficient. Furthermore, much of the DNA does not encode any functional product, and transcription of such sequences would be pointless. Transcription is, in fact, a highly selective process: individual genes are transcribed only as their products are needed. However, this selectivity imposes a fundamental problem on the cell: how to recognize individual genes and transcribe them at the proper time and place. Like replication, transcription requires three major components: 1. A DNA template 2. The raw materials (ribonucleotide triphosphates) needed to build a new RNA molecule 3. The transcription apparatus, consisting of the proteins necessary for catalyzing the synthesis of RNA ## The Template In 1970 , Oscar Miller , Jr . ; Barbara Hamkalo ; and Charles Thomas used electron microscopy to demonstrate that RNA is transcribed from a DNA template. They broke open salamander oocytes , extracted chromatin , and spread the chromatin onto a fine-mesh grid . Under the electron microscope , they observed Christmas-tree-like structures , each consisting of a thin central fiber (the trunk of the tree) to which were attached strings (the branches) bearing granules (Figure 13.3a). The addition of deoxyribonuclease (an enzyme that degrades DNA) caused the central fibers to disappear, indicating that the “tree trunks” were DNA molecules. Ribonuclease (an enzyme that degrades RNA) removed the granular strings, indicating that the branches were RNA. Their conclusion was that each “Christmas tree” represents a gene undergoing transcription (Figure 13.3b). The transcription of each gene begins at the top of the tree ; there , little of the DNA has been transcribed , and the RNA branches are short. As the transcription apparatus moves down the tree , transcribing more of the template , the RNA molecules lengthen , producing the long branches at the bottom. The template for RNA synthesis, as for DNA synthesis, is a single strand of the DNA double helix . Unlike replication , however , the transcription of a gene takes place on only one of the two nucleotide strands of DNA (Figure 13.4). The nucleotide strand used for transcription is termed the template strand. The other strand, called the nontemplate strand, is not ordinarily transcribed. Thus, within a gene, only one of the nucleotide strands is normally transcribed into RNA (although there are some exceptions to this rule). ## The Transcribed Strand During transcription , an RNA molecule that is complementary and antiparallel to the DNA template strand is synthesized (see Figure 13.4). The RNA transcript has the same polarity and base sequence as the nontemplate strand except that it contains U rather than T. In most organisms , each gene is transcribed from a single DNA strand , but different genes may be transcribed from different strands , as shown in Figure 13.5. ## The Transcription Unit A transcription unit is a stretch of DNA that encodes an RNA molecule and the sequences necessary for its transcription. How does the complex of enzymes and proteins that performs transcription-the transcription apparatus-recognize a transcription unit? How does it know which DNA strand to read and where to start and stop? This information is encoded by the DNA sequence. Included within a transcription unit are three critical regions: a promoter, an RNA-coding region, and a terminator (Figure 13.6). The promoter is a DNA sequence that the transcription apparatus recognizes and binds. The promoter indicates which of the two DNA strands is to be read as the template and the direction of transcription. It also determines the transcription start site, the first nucleotide that will be transcribed into RNA. In many transcription units, the promoter is located next to the transcription start site but is not itself transcribed. The second critical region of the transcription unit is the RNA-coding region , a sequence of DNA nucleotides that is copied into an RNA molecule. The third component of the transcription unit is the terminator , a sequence of nucleotides that signals where transcription is to end. Terminators are usually part of the RNA-coding sequence; transcription stops only after the terminator has been copied into RNA. Molecular biologists often use the terms upstream and downstream to refer to the direction of transcription and the locations of nucleotide sequences surrounding the RNA-coding region. The transcription apparatus is said to move downstream during transcription: it binds to the promoter (which is usually upstream of the transcription start site) and moves toward the terminator (which is downstream of the start site). When DNA sequences are written out , often the sequence of only one of the two strands is listed . Molecular biologists typically write the sequence of the nontemplate strand because it will be the same as the sequence of the RNA transcribed from the template strand (with the exception that U in RNA replaces T in DNA). By convention, the sequence of the nontemplate strand is written with the 5 end on the left and the 3 end on the right. The first nucleotide transcribed (the transcription start site) is numbered +1 ; nucleotides downstream of the start site are assigned positive numbers, and nucleotides upstream of the start site are assigned negative numbers. So, nucleotide +34 would be 34 nucleotides downstream of the start site, whereas nucleotide -75 would be 75 nucleotides upstream of the start site. There is no nucleotide numbered 0. ## The Substrate for Transcription RNA is synthesized from ribonucleoside triphosphates (rNTPs), each consisting of a ribose sugar and a base (a nucleoside) attached to three phosphate groups (Figure 13.7). In RNA synthesis, nucleotides are added one at a time to the 3'-OH group of the growing RNA molecule. Two phosphate groups are cleaved from the incoming ribonucleoside triphosphate; the remaining phosphate group participates in a phosphodiester bond that connects the nucleotide to the growing RNA molecule. The overall chemical reaction for the addition of each nucleotide is RNA + rNTP → RNA+1 + PP; where PP; represents pyrophosphate. Nucleotides are always added to the 3' end of the RNA molecule, and the direction of transcription is therefore 5'→3' (Figure 13.8), the same as the direction of DNA synthesis during replication. Thus, the newly synthesized RNA is complementary and antiparallel to the template strand. Unlike DNA synthesis , RNA synthesis does not require a primer. ## The Transcription Apparatus As we have seen, DNA replication requires a number of different enzymes and proteins. Although transcription might initially appear to be quite different because a single enzyme- RNA polymerase-carries out all the required steps, the two processes on closer inspection are actually similar. The action of RNA polymerase is enhanced by a number of accessory proteins that join and leave the polymerase at different stages of the process. Each accessory protein is responsible for providing or regulating a special function. Thus, transcription, like replication, requires an array of proteins. ## Bacterial RNA Polymerase Bacterial cells typically possess only one type of RNA polymerase, which catalyzes the synthesis of all classes of bacterial RNA: mRNA, tRNA, and rRNA. Bacterial RNA polymerase is a large multimeric enzyme (meaning that it consists of several polypeptide chains). At the heart of most bacterial RNA polymerases are five subunits (individual polypeptide chains) that make up the core enzyme: two copies of a subunit called alpha (a) and single copies of subunits beta (β), beta prime (β ), and omega (ω) (Figure 13.9). The ω subunit is not essential for transcription, but it helps stabilize the enzyme. The core enzyme catalyzes the elongation of the RNA molecule by the addition of RNA nucleotides. Other functional subunits join and leave the core enzyme at particular stages of the transcription process. The sigma (σ) factor controls the binding of RNA polymerase to the promoter. Without sigma, RNA polymerase initiates transcription at a random point along the DNA. After sigma has associated with the core enzyme (forming a holoenzyme), RNA polymerase binds stably only to the promoter and initiates transcription at the proper start site. Sigma is required only for promoter binding and initiation; after a few RNA nucleotides have been joined together, sigma usually detaches from the core enzyme. Many bacteria have multiple types of sigma factors; each type initiates the binding of RNA polymerase to a particular set of promoters. ## Eukaryotic RNA Polymerases Most eukaryotic cells possess three distinct types of RNA polymerase, each of which is responsible for transcribing a different class of RNA: RNA polymerase I transcribes rRNA; RNA polymerase II transcribes pre-mRNAs, snoRNAs, some miRNAs, and some snRNAs; and RNA polymerase III transcribes other small RNA molecules-specifically tRNAs, small rRNAs, some miRNAs, and some snRNAs (Table 13.3). RNA polymerases I, II, and III are found in all eukaryotes. Two additional RNA polymerases, RNA polymerase IV transcribes siRNAs that silence transposons and RNA polymerase V transcribes siRNAs that play a role in DNA methylation and chromatin structure. The importance of RNA polymerase is illustrated by α-amanitin, a deadly compound produced by death cap mushrooms. α-Amanitin is a potent inhibitor of RNA polymerase II; it binds to RNA polymerase and jams the moving parts of the enzyme, interfering with its ability to move along the DNA template. In the presence of α-amanitin, RNA synthesis slows from its normal rate of several thousand nucleotides per minute to just a few nucleotides per minute. The results are catastrophic, leading to cell death. The liver, where the toxin accumulates, is irreparably damaged and stops functioning. In severe cases, it causes death. All eukaryotic polymerases are large multimeric enzymes, typically consisting of more than a dozen subunits. Some subunits are common to all RNA polymerases, whereas others are limited to one of the polymerases. As in bacterial cells, a number of accessory proteins bind to the core enzyme and affect its function. ## Bacterial Transcription consists of Initiation, Elongation, and Termination Now that we’ve considered some of the major components of transcription, we’re ready to take a detailed look at the process. Transcription can be conveniently divided into three stages: 1. **Initiation**, in which the transcription apparatus assembles on the promoter and begins the synthesis of RNA 2. **Elongation**, in which DNA is threaded through RNA polymerase and the polymerase unwinds the DNA and adds new nucleotides, one at a time, to the 3’ end of the growing RNA strand 3. **Termination**, the recognition of the end of the transcription unit and the separation of the RNA molecule from the DNA template We first examine each of these steps in bacterial cells, in which the process is best understood; then we consider eukaryotic and archaeal transcription. ## Initiation Initiation comprises all the steps necessary to begin RNA synthesis, including (1) promoter recognition, (2) formation of a transcription bubble, (3) creation of the first bonds between rNTPs, and (4) escape of the transcription apparatus from the promoter. Transcription initiation requires that the transcription apparatus recognize and bind to the promoter. At this step, the selectivity of transcription is enforced: the binding of RNA polymerase to the promoter determines which parts of the DNA template are to be transcribed, and how often. Different genes are transcribed with different frequencies, and promoter binding is important in determining the frequency of transcription for a particular gene. Promoters also have different affinities for RNA polymerase. Even within a single promoter, affinity for RNA polymerase can vary with the passage of time, depending on the promoter’s interaction with RNA polymerase and a number of other factors. ## Bacterial Promoters Essential information for the transcription apparatus-where it will start transcribing, which strand is to be read, and in what direction the RNA polymerase will move-is embedded in the nucleotide sequence of the promoter. In bacterial cells, promoters are usually adjacent to the RNA-coding region. An examination of many promoters in *E. coli* and other bacteria reveals a general feature: although most of the nucleotides at most sites vary among these promoters, short stretches of nucleotides are common to many. Furthermore, the locations of these nucleotides relative to the transcription start site are similar in most promoters. These short stretches of common nucleotides are called consensus sequences. A consensus sequence is the set of the most commonly encountered nucleotides among sequences that possess considerable similarity, or consensus (Figure 13.10). The presence of consensus in a set of nucleotides usually implies that the sequence is associated with an important function. The most commonly encountered consensus sequence, found in almost all bacterial promoters, is centered about 10 bp upstream of the start site. Called the -10 consensus sequence, or sometimes the Pribnow box, this consensus sequence, 5'-TATAAT-3' 3'-ATATTA-5' is often written simply as TATAAT (Figure 13.11). Remember that TATAAT is just the consensus sequence-representing the most commonly encountered nucleotides at each of these sites (see Figure 13.10). In most prokaryotic promoters, the actual sequence is not TATAAT. Another consensus sequence common to most bacterial promoters is TTGACA, which lies approximately 35 nucleotides upstream of the start site and is termed the -35 consensus sequence (see Figure 13.11). The nucleotides on either side of the 10 and -35 consensus sequences and those between them vary greatly from promoter to promoter, suggesting that these nucleotides are not very important in promoter recognition. The function of these consensus sequences in bacterial promoters has been studied by inducing mutations at various positions within the consensus sequences and observing the effect of the changes on transcription. These studies reveal that most base substitutions within the - 10 and - 35 consensus sequences reduce the rate of transcription; these substitutions are termed down mutations because they slow down the rate of transcription. Occasionally, a particular change in a consensus sequence increases the rate of transcription; such a change is called an up mutation. The sigma factor, mentioned earlier, associates with the core RNA polymerase enzyme (Figure 13.12a) to form a holoenzyme, which binds to the -35 and -10 consensus sequences in the DNA promoter (Figure 13.12b). Although it binds only the nucleotides of the consensus sequences, the enzyme extends from -50 to +20 when bound to the promoter. The holoenzyme initially binds weakly to the promoter but then undergoes a change in structure that allows it to bind more tightly and unwind the double-stranded DNA (Figure 13.12c). Unwinding begins within the -10 consensus sequence and extends downstream for about 14 nucleotides, including the start site (from nucleotides - 12 to + 2). Some bacterial promoters contain a third consensus sequence that also takes part in the initiation of transcription. Called the upstream element , this sequence contains a number of A-T pairs and is found at about -40 to -60. A number of proteins may bind to sequences in and near the promoter; some stimulate the rate of transcription and others repress it. We will consider these proteins, which regulate gene expression, in Chapter 16. ## Initial RNA Synthesis Once the holoenzyme has bound to the promoter, RNA polymerase is positioned over the transcription start site (at position +1) and has unwound the DNA to produce a single-stranded template . The orientation and spacing of the consensus sequences on a DNA strand determine which strand will be the template for transcription and thereby determine the direction of transcription. The position of the start site is determined not by the sequences located there but by the locations of the consensus sequences, which position RNA polymerase so that the enzyme’s active site is aligned for the initiation of transcription at +1. If the consensus sequences are artificially moved upstream or downstream, the location of the starting point of transcription correspondingly changes. To begin the synthesis of an RNA molecule, RNA polymerase pairs the base at the start site on the DNA template strand with its complementary base on an rNTP (Figure 13.12d). No primer is required to initiate the synthesis of the 5’ end of the RNA molecule. Two of the three phosphate groups are cleaved from each rNTP as the nucleotide is added to the 3’ end of a growing RNA molecule. However, because the 5’ end of the first rNTP does not take part in the formation of a phosphodiester bond, all three of its phosphate groups remain. An RNA molecule therefore possesses, at least initially, three phosphate groups at its 5’ end (Figure 13.12e). Often in the course of initiation , RNA polymerase repeatedly generates and releases short transcripts , from 2 to 6 nucleotides in length , while still bound to the promoter. This process , termed abortive initiation , occurs in both prokaryotes and eukaryotes. After several abortive initiation attempts , the polymerase synthesizes an RNA molecule from 9 to 12 nucleotides in length , which allows the polymerase to transition to the elongation stage . ## Elongation At the end of initiation, RNA polymerase undergoes a change in its conformation (shape) and thereafter is no longer able to bind to the consensus sequences in the promoter. This change allows the polymerase to escape from the promoter and begin transcribing downstream. The sigma factor is usually released after initiation, although some RNA polymerases may retain sigma throughout elongation. As it moves downstream along the template , RNA polymerase progressively unwinds the DNA at the leading (downstream) edge of the transcription bubble , joining nucleotides to the growing RNA molecule according to the sequence of the template , and rewinds the DNA at the trailing (upstream) edge of the bubble. In bacterial cells at 37°C , about 40 nucleotides are added per second. This rate of RNA synthesis is much lower than that of DNA synthesis , which is 1000 to 2000 nucleotides per second in bacterial cells . ## The Transcription Bubble Transcription takes place within a short stretch of about 18 nucleotides of unwound DNA-the transcription bubble. Within this region, RNA is continuously synthesized. About 8 to 10 nucleotides of newly synthesized RNA are paired with nucleotides on the DNA template at any one time. As the transcription apparatus moves down the template , it generates positive supercoiling ahead of the transcription bubble and negative supercoiling behind it. Topoisomerase enzymes probably relieve the stress associated with the unwinding and rewinding of DNA in transcription, as they do in DNA replication. ## Transcriptional Pausing A number of features of RNA or DNA, such as secondary structures, specific sequences, or the presence of nucleosomes, cause RNA polymerase to pause during the elongation stage of transcription. Such pauses are often caused by backtracking—when the RNA polymerase slides backward along the DNA template strand. Backtracking disengages the 3’-OH group of the RNA molecule from the active site of RNA polymerase and temporarily halts further RNA synthesis. Cells use several mechanisms to minimize backtracking, including proteins that cleave the backtracked RNA in the active site, generating a new 3’-OH group to which new nucleotides can then be added. In bacterial cells, translation of mRNA by ribosomes closely follows transcription (see Chapter 15), and the presence of ribosomes moving along the mRNA in a 5’→3’ direction prevents backtracking of the RNA polymerase at the 3’ end of the mRNA. Backtracking is important in transcriptional proofreading, as we will see shortly. Transitory pauses in transcription are important in the coordination of transcription and translation in bacteria (see the discussion of attenuation in Chapter 16), as well as in the coordination of RNA processing in eukaryotes. Pausing also affects the rate of RNA synthesis. Sometimes a pause may be stabilized by sequences in the DNA that ultimately lead to the termination of transcription. ## Accuracy of Transcription Although RNA polymerase is quite accurate in incorporating nucleotides into the growing RNA chain, errors do occasionally arise. Research has demonstrated that RNA polymerase is capable of a type of proofreading in the course of transcription . When RNA polymerase incorporates a nucleotide that does not match the DNA template , it backtracks and cleaves the last two nucleotides (including the misincorporated nucleotide) from the growing RNA chain. RNA polymerase then proceeds forward, transcribing the DNA template again. ## Termination RNA polymerase adds nucleotides to the 3’ end of the growing RNA molecule until it transcribes a terminator . Most terminators are found upstream of the site at which termination actually takes place . Transcription therefore does not suddenly stop when polymerase reaches a terminator , like a car stopping at a stop sign . Rather , transcription stops after the terminator has been transcribed , like a car that stops only after running over a speed bump . At the terminator , several overlapping events are needed to bring an end to transcription : RNA polymerase must stop synthesizing RNA , the newly made RNA molecule must be released from RNA polymerase , the RNA molecule must dissociate fully from the DNA , and RNA polymerase must detach from the DNA template. Bacterial cells possess two major types of terminators. Rho-dependent terminators are able to cause the termination of transcription only in the presence of an ancillary protein called the rho factor (ρ). Rho-independent terminators (also known as intrinsic terminators) are able to cause the end of transcription in the absence of the rho factor. ## Rho-dependent Terminators Rho-dependent terminators have two features. The first is the DNA sequence of the terminator itself ; this sequence causes the RNA polymerase to pause. The second feature is a DNA sequence upstream of the terminator that encodes a stretch of RNA that is usually rich in cytosine nucleotides and devoid of any secondary structures. This RNA sequence , called the rho utilization (rut) site , serves as a binding site for the rho factor . Once rho binds to the RNA , it moves toward its 3’ end , following the RNA polymerase (Figure 13.13). When RNA polymerase encounters the terminator , it pauses , allowing rho to catch up . The rho factor has helicase activity , which it uses to unwind the DNA-RNA hybrid in the transcription bubble , bringing transcription to an end. ## Rho-independent Terminators Rho-independent terminators, which make up about 50% of all terminators in prokaryotes, have two common features. First , they contain inverted repeats , which are sequences of nucleotides on the same strand that are inverted and complementary . When these inverted repeats are transcribed into RNA and bind to each other , a hairpin forms (Figure 13.14). Second , in rho-independent terminators, a string of seven to nine adenine nucleotides follows the inverted repeat in the template DNA. The transcription of these adenines produces a string of uracil nucleotides after the hairpin in the transcribed RNA. The string of uracils in the RNA molecule causes the RNA polymerase to pause , allowing time for the hairpin structure to form. Evidence suggests that the formation of the hairpin destabilizes the DNA-RNA pairing , causing the RNA molecule to separate from its DNA template. Separation may be facilitated by the adenine-uracil base pairings, which are relatively weak compared with other types of base pairings. When the RNA transcript has separated from the template , RNA synthesis can no longer continue (see Figure 13.14). ## Polycistronic mRNA In bacteria , a group of genes is often transcribed into a single RNA molecule ; such a molecule is termed polycistronic mRNA. Thus , polycistronic mRNA is produced when a single terminator is present at the end of a group of several genes that are transcribed together , instead of each gene having its own terminator. Polycistronic mRNA does occur in some eukaryotes , such as *Caenorhabditis elegans*, but it is uncommon in eukaryotes. You can view the process of transcription , including initiation , elongation , and termination , in Animation 13.1 . The animation shows how the different parts of the transcription unit interact to bring about the complete synthesis of an RNA ## Transcription in Archaea Is More Similar to Transcription in Eukaryotes Than to Transcription in Bacteria Some 2 billion to 3 billion years ago , life diverged into three lines of evolutionary descent : the bacteria (also called eubacteria) , the archaea , and the eukaryotes (see Chapter 2). Although bacteria and archaea are superficially similar-both are unicellular and lack a nucleus-the results of studies of their DNA sequences and other biochemical properties indicate that they are as distantly related to each other as they are to eukaryotes. The evolutionary distinctions between archaea, bacteria, and eukaryotes are clear. But did eukaryotes first diverge from an ancestral prokaryote, with a later separation of prokaryotes into bacteria and archaea, or did the archaea and the bacteria split first, with the eukaryotes later evolving from one of these groups? Studies of transcription in bacteria, archaea, and eukaryotes have yielded important clues to the evolutionary relationships between these organisms. Archaea, like bacteria, have a single RNA polymerase, but this enzyme is most similar to the RNA polymerases of eukaryotes. As discussed earlier, bacterial RNA polymerase consists of 5 subunits, whereas eukaryotic RNA polymerases are much more complex ; RNA polymerase II, for example, is composed of 12 subunits. Archaeal RNA polymerase is similarly complex, with 11 or more subunits. Furthermore, the amino acid sequence of archaeal RNA polymerase is similar to that of eukaryotic RNA polymerase II. Archaeal promoters contain a consensus sequence similar to the TATA box found in eukaryotic promoters. The archaeal TATA box is found approximately 27 bp upstream of the transcription start site and helps to determine the location of the transcription start site, as it does in eukaryotes. Archaea possess a TATA-binding protein (TBP), which is a critical transcription factor for all three of the eukaryotic polymerases but not for bacterial RNA polymerase. TBP binds the TATA box in archaea with the help of another transcription factor, TFIIB, which is also found in eukaryotes but not in bacteria. However, some other regulators of transcription found in archaea are more similar to those found in bacteria, so transcription in archaea is not entirely eukaryotic in nature. As prokaryotes, archaea lack a nuclear membrane, but many species do produce histone proteins, which help compact the DNA and form nucleosome-like structures. Thus, transcription , one of the most basic of life processes , has strong similarities in eukaryotes and archaea, suggesting that these two groups are more closely related to each other than either is to bacteria. This conclusion is supported by other data, including those obtained from a comparison of gene sequences.