H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 46
Текст из файла (страница 46)
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. In prokaryotic DNA the genes areclosely packed with very few noncoding gaps, and the DNAis transcribed directly into colinear mRNA, which then istranslated into protein.This economic clustering of genes devoted to a singlemetabolic function does not occur in eukaryotes, even simpleones like yeasts, which can be metabolically similar to bacteria.
Rather, eukaryotic genes devoted to a single pathwayare most often physically separated in the DNA; indeed suchgenes usually are located on different chromosomes. Eachgene is transcribed from its own promoter, producing onemRNA, which generally is translated to yield a single polypeptide (Figure 4-12b).When researchers first compared the nucleotide sequences of eukaryotic mRNAs from multicellular organismswith the DNA sequences encoding them, they were surprisedto find that the uninterrupted protein-coding sequence of agiven mRNA was broken up (discontinuous) in its corresponding section of DNA.
They concluded that the eukaryotic gene existed in pieces of coding sequence, the exons,separated by non-protein-coding segments, the introns. Thisastonishing finding implied that the long initial primary transcript—the RNA copy of the entire transcribed DNAsequence—had to be clipped apart to remove the introns andthen carefully stitched back together to produce manyeukaryotic mRNAs.Although introns are common in multicellular eukaryotes, they are extremely rare in bacteria and archaea and112CHAPTER 4 • Basic Molecular Genetic Mechanisms(a) Prokaryotes(b) EukaryotesE.
coli genomeYeast chromosomestrp operonEDCTRP1BTRP4IVAkb1550TRP2Start sitefor trp mRNAsynthesis580VTRP5Transcriptiontrp mRNA5910VIITRP33680XIStart sites forprotein synthesisTranscription andRNA processingTranslationtrpmRNAsTranslationEDProteinsCProteinsBA32541▲ FIGURE 4-12 Comparison of gene organization,transcription, and translation in prokaryotes and eukaryotes.(a) The tryptophan (trp) operon is a continuous segment of theE. coli chromosome, containing five genes (blue) that encode theenzymes necessary for the stepwise synthesis of tryptophan.The entire operon is transcribed from one promoter into onelong continuous trp mRNA (red).
Translation of this mRNA beginsat five different start sites, yielding five proteins (green). The orderof the genes in the bacterial genome parallels the sequentialfunction of the encoded proteins in the tryptophan pathway.(b) The five genes encoding the enzymes required for tryptophansynthesis in yeast (Saccharomyces cerevisiae) are carried on fourdifferent chromosomes. Each gene is transcribed from its ownpromoter to yield a primary transcript that is processed into afunctional mRNA encoding a single protein.
The lengths of theyeast chromosomes are given in kilobases (103 bases).uncommon in many unicellular eukaryotes such as baker’syeast. However, introns are present in the DNA of virusesthat infect eukaryotic cells. Indeed, the presence of intronswas first discovered in such viruses, whose DNA is transcribed by host-cell enzymes.cytoplasm before it can be translated into protein.
Thustranscription and translation cannot occur concurrently ineukaryotic cells.All eukaryotic pre-mRNAs initially are modified at thetwo ends, and these modifications are retained in mRNAs.As the 5 end of a nascent RNA chain emerges from the surface of RNA polymerase II, it is immediately acted on byseveral enzymes that together synthesize the 5 cap, a7-methylguanylate that is connected to the terminal nucleotide of the RNA by an unusual 5,5 triphosphate linkage(Figure 4-13). The cap protects an mRNA from enzymaticdegradation and assists in its export to the cytoplasm. Thecap also is bound by a protein factor required to begin translation in the cytoplasm.Processing at the 3 end of a pre-mRNA involves cleavage by an endonuclease to yield a free 3-hydroxyl group towhich a string of adenylic acid residues is added one at a timeby an enzyme called poly(A) polymerase.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
