Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 49
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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. In multicellular organisms, control of geneexpression is largely directed toward assuring that the rightgene is expressed in the right cell at the right time during embryological development and tissue differentiation.
Here wedescribe the basic features of transcription control in bacteria, using the lac operon in E. coli as our primary example.Many of the same processes, as well as others, are involvedin eukaryotic transcription control, which is discussed inChapter 11.In E.
coli, about half the genes are clustered into operons each of which encodes enzymes involved in a particularmetabolic pathway or proteins that interact to form one multisubunit protein. For instance, the trp operon mentionedearlier encodes five enzymes needed in the biosynthesis oftryptophan (see Figure 4-12). Similarly, the lac operon encodes three enzymes required for the metabolism of lactose,a sugar present in milk. Since a bacterial operon is tran-115scribed from one start site into a single mRNA, all the geneswithin an operon are coordinately regulated; that is, they areall activated or repressed to the same extent.Transcription of operons, as well as of isolated genes, iscontrolled by an interplay between RNA polymerase andspecific repressor and activator proteins.
In order to initiatetranscription, however, E. coli RNA polymerase must be associated with one of a small number of (sigma) factors,which function as initiation factors. The most common onein bacterial cells is 70.Initiation of lac Operon TranscriptionCan Be Repressed and ActivatedWhen E. coli is in an environment that lacks lactose, synthesis of lac mRNA is repressed, so that cellular energy isnot wasted synthesizing enzymes the cells cannot use. In anenvironment containing both lactose and glucose, E. colicells preferentially metabolize glucose, the central moleculeof carbohydrate metabolism. Lactose is metabolized at ahigh rate only when lactose is present and glucose is largelydepleted from the medium. This metabolic adjustment isachieved by repressing transcription of the lac operon untillactose is present, and synthesis of only low levels of lacmRNA until the cytosolic concentration of glucose falls tolow levels.
Transcription of the lac operon under differentconditions is controlled by lac repressor and catabolite activator protein (CAP), each of which binds to a specificDNA sequence in the lac transcription-control region (Figure 4-16, top).For transcription of the lac operon to begin, the 70 subunit of the RNA polymerase must bind to the lac promoter,which lies just upstream of the start site. When no lactose ispresent, binding of the lac repressor to a sequence called thelac operator, which overlaps the transcription start site,blocks transcription initiation by the polymerase (Figure4-16a). When lactose is present, it binds to specific bindingsites in each subunit of the tetrameric lac repressor, causing aconformational change in the protein that makes it dissociatefrom the lac operator.
As a result, the polymerase can initiatetranscription of the lac operon. However, when glucose alsois present, the rate of transcription initiation (i.e., the numberof times per minute different polymerase molecules initiatetranscription) is very low, resulting in synthesis of only lowlevels of lac mRNA and the proteins encoded in the lacoperon (Figure 4-16b).Once glucose is depleted from the media and the intracellular glucose concentration falls, E. coli cells respond bysynthesizing cyclic AMP, cAMP (see Figure 3-27b). As theconcentration of cAMP increases, it binds to a site in eachsubunit of the dimeric CAP protein, causing a conformational change that allows the protein to bind to the CAP sitein the lac transcription-control region.
The bound CAPcAMP complex interacts with the polymerase bound to thepromoter, greatly stimulating the rate of transcription initiation. This activation leads to synthesis of high levels of lac116CHAPTER 4 • Basic Molecular Genetic Mechanisms+1 (transcription start site)PromoterlacZOperatorCAP siteE. coli lac transcription-control genesCAPPol-σ70(a)lac repressor− lactoselacZ+ glucose(low cAMP)No mRNA transcriptionlactose(b)+ lactoselacZ+ glucose(low cAMP)(c)+ lactose− glucose(high cAMP)rate of initiation is further reduced by the lac repressor andsubstantially increased by the cAMP-CAP activator.Low transcriptioncAMPlacZHigh transcription▲ FIGURE 4-16 Regulation of transcription from thelac operon of E.
coli. (Top) The transcription-control region,composed of ≈100 base pairs, includes three protein-bindingregions: the CAP site, which binds catabolite activator protein;the lac promoter, which binds the RNA polymerase–70 complex;and the lac operator, which binds lac repressor. The lacZ gene,the first of three genes in the operon, is shown to the right.(a) In the absence of lactose, very little lac mRNA is producedbecause the lac repressor binds to the operator, inhibitingtranscription initiation by RNA polymerase–70. (b) In thepresence of glucose and lactose, lac repressor binds lactoseand dissociates from the operator, allowing RNA polymerase–70to initiate transcription at a low rate. (c) Maximal transcription ofthe lac operon occurs in the presence of lactose and absence ofglucose. In this situation, cAMP increases in response to the lowglucose concentration and forms the CAP-cAMP complex, whichbinds to the CAP site, where it interacts with RNA polymeraseto stimulate the rate of transcription initiation.mRNA and subsequently of the enzymes encoded by the lacoperon (Figure 4-16c).Although the promoters for different E.
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