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The two processes differ, however, in that transcription does not require a primer,it generally involves only short segments of a DNA molecule, andwithin those segments only one of the two DNA strands serves as atemplate. We begin our discussion by introducing the enzymes responsible for transcription.RNA Is Synthesized by RNA PolymerasesThe discovery of DNA polymerase and its dependence on a DNA template encouraged a search for an enzyme that synthesizes an RNAstrand complementary to a DNA template.
Such an enzyme, capable offorming an RNA polymer from ribonucleoside 5'-triphosphates, wasisolated from bacterial extracts in 1959 by four independent researchgroups. This enzyme, DNA-directed RNA polymerase, requires, inaddition to a DNA template, all four ribonucleoside 5'-triphosphates(ATP, GTP, UTP, and CTP) as precursors of the nucleotide units ofRNA, as well as Mg 2+ . The purified enzyme also contains Zn 2+ .
Thefundamental chemistry of RNA synthesis has much in common withDNA synthesis. RNA polymerase elongates an RNA strand by addingribonucleotide units to the 3'-hydroxyl end of the RNA chain and thusbuilds RNA chains in the 5'—>3' direction. The 3'-hydroxyl group actsas nucleophile, attacking at the a-phosphate of the incoming ribonucleoside triphosphate (as illustrated for DNA synthesis in Fig.
24-5) andreleasing pyrophosphate. The overall reaction is(NMP)n + NTPRNA> (NMP) n+1 + PPiLengthenedRNA(25-1)RNA polymerase requires DNA for activity and is most active witha double-stranded DNA as template. Only one of the two DNA strandsis used as a template, copied in the 3'—»5' direction (antiparallel to thenew RNA strand) just as in DNA replication. Each nucleotide in thenewly formed RNA is selected by Watson-Crick base-pairing interac-857858Part IV Information PathwaysFigure 25-1 Transcription by RNA polymerase inE. coli. To synthesize an RNA strand complementary to one of two DNA strands, the DNA is transiently unwound.
Strand designations are summarized in Table 25-1. (a) About 17 base pairs areunwound at any given time. A short RNA-DNAhybrid (about 12 base pairs) is present in the unwound region. The transcription bubble moves fromleft to right as shown, keeping pace with RNA synthesis. The DNA is unwound ahead and rewoundbehind as RNA is transcribed. Arrows show the direction in which the DNA and the RNA-DNA hybrid must rotate to permit this process. As theDNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand is extruded, (b) Supercoiling of DNA brought about by transcription.
Positive supercoils form ahead of the transcriptionbubble and negative supercoils form behind.tions; uridylate (U) residues are inserted in the RNA opposite to adenylate residues in the DNA template, adenylate residues are insertedopposite to thymidylate residues. Guanylate and cytidylate residues inDNA specify cytidylate and guanylate, respectively, in the new RNAstrand.Unlike DNA polymerase, RNA polymerase does not require aprimer to initiate synthesis.
Initiation of RNA synthesis, however, occurs only at specific sequences called promoters (described below).RNA synthesis usually starts with a GTP or ATP residue, whose 5'triphosphate group is not cleaved to release PPj but remains intactthroughout transcription. During transcription the new RNA strandbase-pairs temporarily with the DNA template to form a short lengthof hybrid RNA-DNA double helix, which is essential to the correctreadout of the DNA strand (Fig.
25-1). The RNA in this hybrid duplex"peels off" shortly after its formation.To enable RNA polymerase to synthesize an RNA strand complementary to one of the DNA strands, the DNA duplex must unwind overa short distance, forming a transcription "bubble." During transcription, the E. coli RNA polymerase generally keeps about 17 base pairsunwound, unwinding the DNA ahead and rewinding it behind.
Because the DNA is a helix, this process requires considerable rotation ofthe nucleic acid molecules (Fig. 25-la). Rotation is restricted in mostDNAs by DNA-binding proteins and other structural barriers, and amoving RNA polymerase generates waves of positive supercoils aheadof and negative supercoils behind the point at which transcription isoccurring (Fig. 25-lb). This transcription-driven supercoiling of DNAhas been observed both in vitro and, in bacteria, in vivo. In the cell, theRNA polymerase RewindingTranscription bubbleNontemplate(coding)strandUnwindingDNARNA-DNA hybridDirection of transcription,(a)PositivesupercoilsNegativesupercoilsDirection of transcriptionRNA(b)Chapter 25 RNA MetabolismRNA transcriptsDNA36 x 103 bptopological problems caused by transcription are relieved through theaction of topoisomerases.
Once begun, transcription inE. coli proceedsat a rate of about 50 nucleotides per second.The sequences of two complementary DNA strands are different,and the two strands serve different functions in transcription. A variety of designations are used to distinguish the two strands (Table25-1). The strand that serves as template for RNA synthesis is calledthe template strand or minus (-) strand. In any chromosome, different genes may use different strands as template (Fig.
25-2). The DNAstrand complementary to the template is called the nontemplatestrand or plus ( + ) strand. It is identical in base sequence with theRNA transcribed from the gene, with U in place of T (Fig. 25-3). Thenontemplate strand is also sometimes called the coding strand, eventhough it has no direct function in either transcription or protein synthesis. The regulatory sequences needed for transcription (describedlater in this chapter) are by convention given as sequences in thenontemplate (or coding or +) strand.(5')CGCTATAGCGTTT(3')(3') GCGATATCGCAAA(5')DNA nontemplate ( + ) strandDNA template (-) strand(5') CGCUAUAGCGUUU(3')RNA transcript859Figure 25-2 The genetic information of the adenovirus is encoded by a double-stranded DNA molecule (36,000 base pairs), both strands of which encode proteins.
The information for most proteinsis encoded by the top strand (transcribed left toright), but some is encoded by the bottom strandand is transcribed in the opposite direction. Synthesis of mRNAs in adenovirus is actually much morecomplex than shown here. Many of the mRNAsshown for the upper strand are initially synthesized as one long transcript derived from morethan two-thirds of the length of the DNA. Thetranscript is extensively processed to produce themRNAs for most of the individual gene products.Adenovirus causes some types of upper respiratorytract infections in some vertebrates.Table 25-1 Alternative designations forDNA strands in transcriptionTemplate strandMinus ( —) strandNontemplate strandPlus ( + ) strandCoding strandFigure 25-3 The two complementary strands ofDNA are defined by their function in transcription.The RNA transcript is synthesized on the complementary template (-) strand, and it is identical insequence (with U in place of T) to the nontemplate( + ) or coding strand.E.
coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA. It is a large (Mr 390,000) and complex enzyme,containing five core subunits and a sixth subunit, called a or a70 (Mr70,000), that binds transiently to the core and directs the enzyme tospecific initiation sites on the DNA (described below). These six subunits constitute the RNA polymerase holoenzyme (Fig. 25-4). RNApolymerases, whether from E. coli or other organisms, lack a proofreading 3'—»5' exonuclease activity such as that found in many DNApolymerases. As a result, during transcription about one error is madefor every 104 to 105 ribonucleotides incorporated into RNA.
Given thatmany copies of an RNA are generally produced from a single gene andthat all of the RNAs are eventually degraded and replaced, a rare mistake in an RNA molecule is of less consequence to the cell than a mistake in the permanent information stored in DNA.Figure 25-4 The subunit structure of E. coliRNA polymerase. The a (of which there are two),P, ft', (o, and a subunits have molecular weights of36,500, 151,000, 155,000, 11,000, and 70,000, respectively. The a subunit is also called a70. The catalytic site for RNA synthesis is believed to be in theP subunit.SubunitCore enzyme860Part IV Information PathwaysRNA Synthesis Is Initiated at PromotersInitiation of RNA synthesis at random points in a DNA molecule wouldbe an extraordinarily wasteful process.
Instead, the RNA polymerasebinds to specific sequences in the DNA called promoters, which directthe transcription of adjacent segments of DNA (genes). The sequencesadjacent to genes where RNA polymerases must bind can be quite variable, and much research has focused on identifying the sequences thatare critical to promoter function. Analysis and comparison of sequences in many different bacterial promoters have revealed similarities in two short sequences located about 10 and 35 base pairs awayfrom the point where RNA synthesis is initiated (Fig. 25-5).
By convention the base pair that begins an RNA molecule is given the number + 1, so these sequences are commonly called the -10 and - 3 5 regions. The sequences are not identical for all bacterial promoters, butcertain nucleotides are found much more often than others at eachposition. The most common nucleotides form what is called a consensus sequence (recall the consensus sequences of oriC in the E. colichromosome; see Fig. 24-10). For most promoters in E. coli and relatedbacteria, the consensus sequence for the -10 region (also called thePribnow box) is (5')TATAAT(3'), and the consensus sequence at the- 3 5 region is (5')TTGACA(3'XFigure 25-5 The sequences of five E.
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