Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 47
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The DNAmolecules of small viruses contain only a few genes,whereas the single DNA molecule in each of the chromosomes of higher animals and plants may contain severalthousand genes.During synthesis of RNA, the four-base language of DNAcontaining A, G, C, and T is simply copied, or transcribed,into the four-base language of RNA, which is identical exceptthat U replaces T. In contrast, during protein synthesis thefour-base language of DNA and RNA is translated into the20–amino acid language of proteins.
In this section we focuson formation of functional mRNAs from protein-codinggenes (see Figure 4-1, step 1 ). A similar process yields theprecursors of rRNAs and tRNAs encoded by rRNA andtRNA genes; these precursors are then further modified toyield functional rRNAs and tRNAs (Chapter 12).A Template DNA Strand Is Transcribed intoa Complementary RNA Chain by RNA PolymeraseDuring transcription of DNA, one DNA strand acts as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form acomplementary RNA chain.
Bases in the template DNAstrand base-pair with complementary incoming rNTPs,which then are joined in a polymerization reaction catalyzedby RNA polymerase. Polymerization involves a nucleophilicattack by the 3 oxygen in the growing RNA chain on the phosphate of the next nucleotide precursor to be added, resulting in formation of a phosphodiester bond and releaseof pyrophosphate (PPi).
As a consequence of this mechanism,RNA molecules are always synthesized in the 5n3 direction (Figure 4-9).The energetics of the polymerization reaction strongly favors addition of ribonucleotides to the growing RNA chainbecause the high-energy bond between the and phosphate of rNTP monomers is replaced by the lower-energyphosphodiester bond between nucleotides. The equilibriumfor the reaction is driven further toward chain elongation bypyrophosphatase, an enzyme that catalyzes cleavage of thereleased PPi into two molecules of inorganic phosphate. Likethe two strands in DNA, the template DNA strand and thegrowing RNA strand that is base-paired to it have opposite5n3 directionality.By convention, the site at which RNA polymerase beginstranscription is numbered 1.
Downstream denotes the direction in which a template DNA strand is transcribed (ormRNA translated); thus a downstream sequence is towardthe 3 end relative to the start site, considering the DNAstrand with the same polarity as the transcribed RNA. Upstream denotes the opposite direction. Nucleotide positionsin the DNA sequence downstream from a start site are indicated by a positive () sign; those upstream, by a negative() sign.353 RNAstrand growthBaseOBaseO5HHOHOHH−OBasestrandOOBaseODNAtemplatePHHOHOHH−OBasePOOBaseOHHH3 HOHOHPolymerizationBaseOBaseOOHHOHHOHHPαO−OOPβO−OOPγO−O−Incoming rNTPBaseBase5▲ FIGURE 4-9 Polymerization of ribonucleotides by RNApolymerase during transcription. The ribonucleotide to beadded at the 3 end of a growing RNA strand is specified bybase pairing between the next base in the template DNA strandand the complementary incoming ribonucleoside triphosphate(rNTP).
A phosphodiester bond is formed when RNA polymerasecatalyzes a reaction between the 3 O of the growing strand andthe phosphate of a correctly base-paired rNTP. RNA strandsalways are synthesized in the 5n3 direction and are oppositein polarity to their template DNA strands.Stages in Transcription To carry out transcription, RNApolymerase performs several distinct functions, as depictedin Figure 4-10. During transcription initiation, RNA polymerase recognizes and binds to a specific site, called a promoter, in double-stranded DNA (step 1). Nuclear RNACHAPTER 4 • Basic Molecular Genetic MechanismsRNA polymeraseINITIATION1 Polymerase binds topromoter sequencein duplex DNA."Closed complex"Start siteon templatestrand110 FIGURE 4-10 Three stages inStop siteon templatestrand5353PromoterFocus Animation: Basic Transcriptional MechanismMEDIA CONNECTIONS2 Polymerase meltsduplex DNA neartranscription start site,forming a transcriptionbubble.
"Opencomplex"3 Polymerase catalyzesphosphodiester linkageof two initial rNTPs.5353Transcriptionbubbletranscription. During initiation of transcription,RNA polymerase forms a transcription bubbleand begins polymerization of ribonucleotides(rNTPs) at the start site, which is locatedwithin the promoter region. Once a DNAregion has been transcribed, the separatedstrands reassociate into a double helix,displacing the nascent RNA except at its 3end. The 5’ end of the RNA strand exits theRNA polymerase through a channel in theenzyme. Termination occurs when thepolymerase encounters a specific terminationsequence (stop site).
See the text for details.Initial rNTPs53534 Polymerase advances35 down templatestrand, melting duplexDNA and adding rNTPsto growing RNA.5353TERMINATION53ELONGATION5 At transcription stop site,polymerase releasescompleted RNA anddissociates from DNA.NascentRNADNA-RNAhybrid region53535CompletedRNA strandpolymerases require various protein factors, called generaltranscription factors, to help them locate promoters and initiate transcription. After binding to a promoter, RNA polymerase melts the DNA strands in order to make the bases inthe template strand available for base pairing with the basesof the ribonucleoside triphosphates that it will polymerize together.
Cellular RNA polymerases melt approximately 14base pairs of DNA around the transcription start site, whichis located on the template strand within the promoter region(step 2 ). Transcription initiation is considered completewhen the first two ribonucleotides of an RNA chain arelinked by a phosphodiester bond (step 3 ).After several ribonucleotides have been polymerized,RNA polymerase dissociates from the promoter DNA andgeneral transcription factors.
During the stage of strand elongation, RNA polymerase moves along the template DNA onebase at a time, opening the double-stranded DNA in front ofits direction of movement and hybridizing the strands behindit (Figure 4-10, step 4 ). One ribonucleotide at a time is addedto the 3 end of the growing (nascent) RNA chain duringstrand elongation by the polymerase. The enzyme maintainsa melted region of approximately 14 base pairs, called thetranscription bubble. Approximately eight nucleotides at the3 end of the growing RNA strand remain base-paired to thetemplate DNA strand in the transcription bubble.
The elongation complex, comprising RNA polymerase, templateDNA, and the growing (nascent) RNA strand, is extraordinarily stable. For example, RNA polymerase transcribes thelongest known mammalian genes, containing ≈2 106 basepairs, without dissociating from the DNA template or releasing the nascent RNA.
Since RNA synthesis occurs at a rate ofabout 1000 nucleotides per minute at 37 C, the elongationcomplex must remain intact for more than 24 hours to assurecontinuous RNA synthesis.During transcription termination, the final stage in RNAsynthesis, the completed RNA molecule, or primary transcript,4.2 • Transcription of Protein-Coding Genes and Formation of Functional mRNA111is released from the RNA polymerase and the polymerasedissociates from the template DNA (Figure 4-10, step 5 ).Specific sequences in the template DNA signal the boundRNA polymerase to terminate transcription. Once released,an RNA polymerase is free to transcribe the same gene againor another gene.grams of the transcription process generally show RNA polymerase bound to an unbent DNA molecule, as in Figure4-10.
However, according to a current model of the interaction between bacterial RNA polymerase and promoter DNA,the DNA bends sharply following its entry into the enzyme(Figure 4-11).Structure of RNA Polymerases The RNA polymerases ofOrganization of Genes Differs in Prokaryoticand Eukaryotic DNAbacteria, archaea, and eukaryotic cells are fundamentallysimilar in structure and function. Bacterial RNA polymerasesare composed of two related large subunits ( and ), twocopies of a smaller subunit (), and one copy of a fifth subunit () that is not essential for transcription or cell viability but stabilizes the enzyme and assists in the assembly ofits subunits.
Archaeal and eukaryotic RNA polymerases haveseveral additional small subunits associated with this corecomplex, which we describe in Chapter 11. Schematic dia-ω subunit−30β subunit−20α subunit−10+10+20β subunit▲ FIGURE 4-11 Current model of bacterial RNApolymerase bound to a promoter. This structure correspondsto the polymerase molecule as schematically shown in step 2 ofFigure 4-10. The subunit is in orange; is in green.
Part ofone of the two subunits can be seen in light blue; the subunit is in gray. The DNA template and nontemplate strandsare shown, respectively, as gray and pink ribbons. A Mg2 ionat the active center is shown as a gray sphere. Numbers indicatepositions in the DNA sequence relative to the transcription startsite, with positive () numbers in the direction of transcriptionand negative () numbers in the opposite direction. [Courtesy ofR. H. Ebright, Waksman Institute.]Having outlined the process of transcription, we now brieflyconsider the large-scale arrangement of information in DNAand how this arrangement dictates the requirements forRNA synthesis so that information transfer goes smoothly. Inrecent years, sequencing of the entire genomes from severalorganisms has revealed not only large variations in the number of protein-coding genes but also differences in their organization in prokaryotes and eukaryotes.The most common arrangement of protein-coding genesin all prokaryotes has a powerful and appealing logic: genesdevoted to a single metabolic goal, say, the synthesis of theamino acid tryptophan, are most often found in a contiguousarray in the DNA.
Such an arrangement of genes in a functional group is called an operon, because it operates as a unitfrom a single promoter. Transcription of an operon producesa continuous strand of mRNA that carries the message for arelated series of proteins (Figure 4-12a). Each section of themRNA represents the unit (or gene) that encodes one of theproteins in the series.
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