Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 48
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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. The resultingpoly(A) tail contains 100–250 bases, being shorter in yeastsand invertebrates than in vertebrates.
Poly(A) polymerase isEukaryotic Precursor mRNAs Are Processedto Form Functional mRNAsIn prokaryotic cells, which have no nuclei, translation of anmRNA into protein can begin from the 5 end of the mRNAeven while the 3 end is still being synthesized by RNA polymerase. In other words, transcription and translation canoccur concurrently in prokaryotes. In eukaryotic cells, however, not only is the nucleus separated from the cytoplasmwhere translation occurs, but also the primary transcripts ofprotein-coding genes are precursor mRNAs (pre-mRNAs)that must undergo several modifications, collectively termedRNA processing, to yield a functional mRNA (see Figure4-1, step 2 ).
This mRNA then must be exported to the1134.2 • Transcription of Protein-Coding Genes and Formation of Functional mRNA13132105106147Start site forRNA synthesisPrimary 5RNAtranscriptPoly(A)site33 cleavage andaddition ofpoly(A) tailExon(A)nPoly(A)tailIntronUTR6HN12354OOHOPOOPON547897-MethylguanylateHβ-GlobinmRNAH32OHOHOOOPO5CH24HOBase 1HHOOO POCH2HOP1H23CH3OO1147(A)n15 linkage5Intron excision,exon ligationNOCH2(A)nNH2Nm7GpppCH3O▲ FIGURE 4-14 Overview of RNA processing toproduce functional mRNA in eukaryotes. The -globingene contains three protein-coding exons (coding region, red)and two intervening noncoding introns (blue). The intronsinterrupt the protein-coding sequence between the codonsfor amino acids 31 and 32 and 105 and 106.
Transcription ofeukaryotic protein-coding genes starts before the sequencethat encodes the first amino acid and extends beyond thesequence encoding the last amino acid, resulting in noncodingregions (gray) at the ends of the primary transcript. Theseuntranslated regions (UTRs) are retained during processing.The 5 cap (m7Gppp) is added during formation of the primaryRNA transcript, which extends beyond the poly(A) site. Aftercleavage at the poly(A) site and addition of multiple A residuesto the 3 end, splicing removes the introns and joins theexons. The small numbers refer to positions in the 147–aminoacid sequence of -globin.Base 2HHOOOHCH3▲ FIGURE 4-13 Structure of the 5 methylated cap ofeukaryotic mRNA.
The distinguishing chemical features are the5n5 linkage of 7-methylguanylate to the initial nucleotide ofthe mRNA molecule and the methyl group on the 2 hydroxylof the ribose of the first nucleotide (base 1). Both these featuresoccur in all animal cells and in cells of higher plants; yeasts lackthe methyl group on nucleotide 1. The ribose of the secondnucleotide (base 2) also is methylated in vertebrates. [SeeA. J.
Shatkin, 1976, Cell 9:645.]untranslated regions (UTRs), at each end. In mammalianmRNAs, the 5 UTR may be a hundred or more nucleotideslong, and the 3 UTR may be several kilobases in length.Prokaryotic mRNAs also usually have 5 and 3 UTRs, butthese are much shorter than those in eukaryotic mRNAs,generally containing fewer than 10 nucleotides.Alternative RNA Splicing Increases the Numberof Proteins Expressed from a SingleEukaryotic GeneIn contrast to bacterial and archaeal genes, the vast majority of genes in higher, multicellular eukaryotes contain multiple introns.
As noted in Chapter 3, many proteins fromMEDIA CONNECTIONSβ-GlobingenomicDNAOverview Animation: Life Cycle of an mRNApart of a complex of proteins that can locate and cleave atranscript at a specific site and then add the correct numberof A residues, in a process that does not require a template.The final step in the processing of many different eukaryotic mRNA molecules is RNA splicing: the internalcleavage of a transcript to excise the introns, followed by ligation of the coding exons. Figure 4-14 summarizes the basicsteps in eukaryotic mRNA processing, using the -globingene as an example.
We examine the cellular machinery forcarrying out processing of mRNA, as well as tRNA andrRNA, in Chapter 12.The functional eukaryotic mRNAs produced by RNAprocessing retain noncoding regions, referred to as 5 and 3114CHAPTER 4 • Basic Molecular Genetic MechanismsEIIIBFibronectin geneFibroblastfibronectin mRNA5Hepatocytefibronectin mRNA5▲ FIGURE 4-15 Cell type–specific splicing of fibronectinpre-mRNA in fibroblasts and hepatocytes. The ≈75-kbfibronectin gene (top) contains multiple exons. The EIIIB andEIIIA exons (green) encode binding domains for specific proteinson the surface of fibroblasts.
The fibronectin mRNA produced inhigher eukaryotes have a multidomain tertiary structure (seeFigure 3-8). Individual repeated protein domains often areencoded by one exon or a small number of exons that codefor identical or nearly identical amino acid sequences. Suchrepeated exons are thought to have evolved by the accidentalmultiple duplication of a length of DNA lying between twosites in adjacent introns, resulting in insertion of a string ofrepeated exons, separated by introns, between the originaltwo introns.
The presence of multiple introns in many eukaryotic genes permits expression of multiple, related proteins from a single gene by means of alternative splicing. Inhigher eukaryotes, alternative splicing is an important mechanism for production of different forms of a protein, calledisoforms, by different types of cells.Fibronectin, a multidomain extracellular adhesive protein found in mammals, provides a good example of alternative splicing (Figure 4-15). The fibronectin gene containsnumerous exons, grouped into several regions corresponding to specific domains of the protein. Fibroblasts produce fibronectin mRNAs that contain exons EIIIA andEIIIB; these exons encode amino acid sequences that bindtightly to proteins in the fibroblast plasma membrane.Consequently, this fibronectin isoform adheres fibroblaststo the extracellular matrix.
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