H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 47
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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. Alternative splicing of the fibronectin primary transcript in hepatocytes, the majortype of cell in the liver, yields mRNAs that lack the EIIIAand EIIIB exons. As a result, the fibronectin secreted byhepatocytes into the blood does not adhere tightly to fibroblasts or most other cell types, allowing it to circulate.During formation of blood clots, however, the fibrinbinding domains of hepatocyte fibronectin binds to fibrin,one of the principal constituents of clots.
The bound fibronectin then interacts with integrins on the membranesof passing, activated platelets, thereby expanding the clotby addition of platelets.More than 20 different isoforms of fibronectin have beenidentified, each encoded by a different, alternatively splicedmRNA composed of a unique combination of fibronectingene exons.
Recent sequencing of large numbers of mRNAsEIIIA33fibroblasts includes the EIIIA and EIIIB exons, whereas theseexons are spliced out of fibronectin mRNA in hepatocytes. Inthis diagram, introns (black lines) are not drawn to scale; mostof them are much longer than any of the exons.isolated from various tissues and comparison of their sequences with genomic DNA has revealed that nearly 60 percent of all human genes are expressed as alternatively splicedmRNAs. Clearly, alternative RNA splicing greatly expandsthe number of proteins encoded by the genomes of higher,multicellular organisms.KEY CONCEPTS OF SECTION 4.2Transcription of Protein-Coding Genes and Formationof Functional mRNATranscription of DNA is carried out by RNA polymerase, which adds one ribonucleotide at a time to the 3end of a growing RNA chain (see Figure 4-10).
The sequence of the template DNA strand determines the orderin which ribonucleotides are polymerized to form an RNAchain.■During transcription initiation, RNA polymerase bindsto a specific site in DNA (the promoter), locally melts thedouble-stranded DNA to reveal the unpaired templatestrand, and polymerizes the first two nucleotides.■During strand elongation, RNA polymerase moves alongthe DNA, melting sequential segments of the DNA andadding nucleotides to the growing RNA strand.■When RNA polymerase reaches a termination sequencein the DNA, the enzyme stops transcription, leading to release of the completed RNA and dissociation of the enzyme from the template DNA.■In prokaryotic DNA, several protein-coding genes commonly are clustered into a functional region, an operon,which is transcribed from a single promoter into onemRNA encoding multiple proteins with related functions(see Figure 4-12a).
Translation of a bacterial mRNA canbegin before synthesis of the mRNA is complete.■In eukaryotic DNA, each protein-coding gene is transcribed from its own promoter. The initial primary tran-■4.3 • Control of Gene Expression in Prokaryotesscript very often contains noncoding regions (introns) interspersed among coding regions (exons).Eukaryotic primary transcripts must undergo RNA processing to yield functional RNAs.
During processing, theends of nearly all primary transcripts from protein-codinggenes are modified by addition of a 5 cap and 3 poly(A)tail. Transcripts from genes containing introns undergosplicing, the removal of the introns and joining of the exons (see Figure 4-14).■The individual domains of multidomain proteins foundin higher eukaryotes are often encoded by individual exons or a small number of exons. Distinct isoforms of suchproteins often are expressed in specific cell types as the result of alternative splicing of exons.■4.3 Control of Gene Expressionin ProkaryotesSince the structure and function of a cell are determined bythe proteins it contains, the control of gene expression is afundamental aspect of molecular cell biology.
Most commonly, the “decision” to initiate transcription of the gene encoding a particular protein is the major mechanism forcontrolling production of the encoded protein in a cell. Bycontrolling transcription initiation, a cell can regulate whichproteins it produces and how rapidly. When transcription ofa gene is repressed, the corresponding mRNA and encodedprotein or proteins are synthesized at low rates. Conversely,when transcription of a gene is activated, both the mRNAand encoded protein or proteins are produced at muchhigher rates.In most bacteria and other single-celled organisms, geneexpression is highly regulated in order to adjust the cell’s enzymatic machinery and structural components to changes inthe nutritional and physical environment. Thus, at any giventime, a bacterial cell normally synthesizes only those proteinsof its entire proteome required for survival under the particular conditions.
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