9 Геном, плазмиды, вирусы (Лекции), страница 8

25-14). An unusual branched lariat structure is formed as anintermediate.Attempts to identify the enzymes that promote splicing of group Iand group II introns produced a major surprise; many of these intronsare self-splicing—no protein enzymes are involved. This was first revealed in studies of the splicing mechanism of the group I rRNA intronfrom the ciliated protozoan Tetrahymena thermophila by Thomas Cechand colleagues in 1982.

These workers proved that no proteins wereinvolved by transcribing Tetrahymena DNA (including the intron) inPrimarytranscriptFigure 25-12 A transesteriflcation reaction. Thisis the first step in the splicing of group I introns.Here, the 3' OH of a guanosine molecule acts asnucleophile.5'-5' exon-UpA3' exonGpU-•3'The 3' OH of guanosineacts as a nucleophile,attacking the phosphate atthe 5' splice site.pG-OHpGpAIntermediate5'--U-OHGpUThe3'OHofthe5'exonbecomes the nucleophile,completing the reaction.5' pGpASpliced RNAFigure 25-13 Splicing mechanism of group Iintrons. The nucleophile in the first step may beguanosine, GMP, GDP, or GTP.5'--UpU-•3'G-OH 3'Chapter 25 RNA MetabolismFigure 25—14 Splicing mechanism of group IIintrons.

The chemistry is similar to that of group Iintron splicing, except for the nucleophile in thefirst step and the novel lariatlike intermediate withone branch having a 2',5'-phosphodiester bond.OHPrimarytranscript•UpGThe 2' OH of a specific adenosinein the intron acts as a nucleophile,attacking the 5' splice site to forma lariat structure.Intermediate-3'pU-2!, 5'-Phosphodiester bondTo 3' end5'The 3' OH of the 51 exon actsas a nucleophile, completingthe reaction.Adenosine in the lariatstructure has threephosphodiester bonds.Spliced RNA51-UpU-8693' +PAOH(3')vitro using bacterial RNA polymerase. The resulting RNA spliced itselfaccurately even though it had never been in contact with any enzymesfrom Tetrahymena. The realization that RNAs, as well as proteins,could have catalytic functions was a milestone in thinking about biological systems.

RNA catalysts are discussed in more detail later inthis chapter.The third and largest group of introns, found in nuclear mRNAprimary transcripts, undergo splicing by the same lariat-formationmechanism as the group II introns. However, they are not self-splicing.Splicing requires the action of specialized RNA-protein complexescontaining a class of eukaryotic RNAs called small nuclear RNAs(snRNAs). Five snRNAs, Ul, U2, U4, U5, and U6, are involved insplicing reactions.

They are found in abundance in the nuclei of manyeukaryotes, range in size from 106 (U6) to 189 (U2) nucleotides, and arecomplexed with proteins to form particles called small nuclear ribonucleqproteins (snRNPs, often referred to as "snurps"). The RNAs andproteins in snRNPs are highly conserved among vertebrates and insects.

Small nuclear RNAs similar to these are also found in yeast andslime molds.Ul snRNA3'C G G U C C A if J C AmUmA5'PPP5'm7G (5')AGGU AAGUt5' Splice siteIntron2'OH\ ^Bulged AExonsAC AUCAUU2 snRNA5'kFigure 25-15 Splicing mechanism in mRNA primary transcripts. The splice sites that mark theintron-exon boundaries of many eukaryotic mRNAshave some conserved sequences. The Ul snRNA hasa sequence near its 5' end complementary to thesplice site at the 5' end of the intron. Base pairingof Ul to this region of the primary transcript helpsdefine the 5' splice site, ip represents pseudouridine(see Fig. 25-25), and "m" indicates methylated residues.

Base pairing of U2 snRNA to the branch sitedisplaces (bulges) and perhaps activates the adenosine, whose 2' OH forms the lariat structurethrough a 2',5'-phosphodiester bond.The Ul snRNA has a sequence complementary to sequences nearthe 5' splice site of nuclear mRNA introns (Fig. 25-15), and the UlsnRNP binds to this region in the primary transcript. Addition of theU2, U4, U5, and U6 snRNPs leads to formation of a complex called the"spliceosome" within which the actual splicing reaction occurs. ATP isrequired for assembly of the spliceosome, but there is no reason tobelieve that the splicing reactions require ATP.The fourth class of intron, found in certain tRNAs, is distinguishedfrom the group I and II introns in that its splicing requires ATP.

In thiscase, a splicing endonuclease cleaves the phosphodiester bonds at bothends of the intron, and the two exons are joined as shown in Figure25-16. The joining reaction is similar to the DNA ligase mechanism(see Fig. 24-15).Introns are not limited to eukaryotes.

Although very rare, severalgenes with introns have now been found in bacteria and bacterial viruses. Bacteriophage T4, for example, has several genes with group Iintrons. Introns appear to be more common in archaebacteria (p. 25)than in E. coli.Figure 25-16 (Facing page) The splicing of yeasttRNA. This splicing pathway requires a highenergy cofactor (ATP) for the ligation step, (a) Theintron is first removed by endonuclease-catalyzedcleavage at both ends, (b) The 2',3'-cyclic phosphate on the 5' exon is cleaved by a cyclic nucleotide phosphodiesterase, leaving a 2' phosphate,(c) The 5' OH left on the 3' exon is then activatedin two steps, (d) The free 3' hydroxyl of the 5' exonacts as a nucleophile to displace AMP, joining thetwo exons with a 3',5'-phosphodiester bond, (e) The2' phosphate is removed to yield the final product.Eukaryotic mRNAs Undergo Additional ProcessingIn eukaryotes, mature mRNAs have distinctive structural features atboth ends.

Most have a 5' cap, a residue of 7-methylguanosine linkedto the 5'-terminal residue of the mRNA through an unusual 5',5'triphosphate linkage (Fig. 25-17). At the 3' end, most eukaryoticmRNAs have a "tail" of 20 to 250 adenylate residues, called thepoly (A) tail. The functions of the 5' cap and the 3' poly(A) tail are onlypartially known. The 5' cap binds to a protein and may participate inthe binding of the mRNA to the ribosome to initiate translation (Chapter 26).

The poly(A) tail also is bound by a specific protein. It is likelythat the 5' cap and poly(A) tail and their associated proteins help protect the mRNA from enzymatic destruction.870Chapter 27 Regulation of Gene Expression963E. coli chromosomepolBdinBuvrBuvrAsul A(b)Damaged DNAsingle-strand gapumuC,DrecAlexA<- — Replication—-•polBdinBuvrBsulArepressor 0.activatedSingle-stranded DNARecA complex ^umuC,DlexADuring induction of the SOS response, RecA protein also cleavesand thus inactivates the repressors that allow bacteriophage A andrelated bacterial viruses to be propagated in a dormant (lysogenic)state within a bacterial host.

These repressors have apparentlyevolved to mimic the LexA repressor, and they are also cleaved at aspecific Ala-Gly peptide bond. This leads to replication of the virusand lysis of the cell to release the new virus particles, permitting thebacteriophage to make a hasty exit from a bacterial cell that is in distress, as described below.Bacteriophage A Provides an Example of aRegulated Developmental SwitchThe objective of regulation of bacterial virus (bacteriophage) genes isusually the orderly assembly of new phage particles without destroying the host cell too soon. The well-studied bacteriophage A provides anexample of a complex and elegant regulatory circuit that determinesthe developmental fate of the virus in a given bacterial cell. This provides a paradigm for the complex problem of development in multicellular organisms.Figure 27-24 The SOS response in E.

coli. SeeTable 24-6 for a description of the functions ofthese genes. The LexA protein is the repressor inthis system, with an operator site (indicated in red)near each gene, (a) The recA gene is not entirelyrepressed by the LexA repressor, and about 1,000RecA protein monomers are normally found in thecell, (b) When DNA is extensively damaged (e.g.,by UV light), DNA replication is halted and thenumber of single-strand gaps in the DNA increases.(c) RecA protein binds to this single-stranded DNA,activating the protein's coprotease activity.(d) While bound to DNA the RecA protein facilitates the cleavage and inactivation of the LexA repressor. When the repressor is inactivated, the SOSgenes, including recA, are induced.

RecA proteinlevels increase 50- to 100-fold.964Part IV Information PathwaysFigure 27-25 Bacteriophage A. (a) Electron micrograph of bacteriophage A virus particles attached toan E. coli cell, (b) Two alternative fates for a bacteriophage A infection: lysis or lysogeny. Under certain conditions (e.g., when the SOS response is induced), the lysogenic state is interrupted and thecell undergoes a lytic cycle (dashed arrow).Lysis or Lysogeny: Two Possible Fates Bacteriophage A (Fig. 27-25)is a medium-sized DNA phage with a chromosome containing 48,502base pairs.

It is a temperate phage, meaning that phage infectiondoes not always result in the destruction of the host cell. The DNA ofthe invading phage has two possible fates (Fig. 27-25b): reproductionleading to generation of new phage particles and lysis of the cell (lyticBacteriophageLysogeniccycleA DNA integrated intohost chromosomeAssembled%bacteriophage A \Chapter 27 Regulation of Gene Expressioncycle) or a relatively benign integration into the host chromosomewhere it can be replicated passively with the host DNA for many generations (lysogeny). The choice between lysis and lysogeny is governed largely by the interactions of five regulatory proteins called CI,CII, Cro, N, and Q.

These proteins regulate transcription from a number of promoters in a regulatory region of the phage DNA describedbelow. The functions of the proteins and promoters are summarized inTable 27-1.Table 27-1 Regulatory elements of bacteriophage ARegulatoryelementProteinsCIFunctionAt low concentrations a repressor of PR and PL and anactivator of PRM; at high concentrations also repressesPRMCIICroNQAn activator of PRE and P intAt low concentrations a repressor of PRM; at highconcentrations also represses PL and PRAn antiterminator at tLlt t Rb tR2j and other terminatorsAn antiterminator for late gene transcriptionPromotersPRMajor rightward transcriptionPLMajor leftward transcriptionPRMTranscription for repressor maintenancePRETranscription for repressor establishmentP intTranscription of genes for integration and excisionThe CI, CII, and Cro proteins are approximately analogous to thetypes of regulatory proteins already described.