8 Регуляция экспрессии генов. Система передачи сигнала (1160077), страница 9
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The molecular basis forthis discrimination has been the subject of intensive investigation. Ageneral conclusion is that regulatory proteins usually have discreteDNA-binding domains. In addition, the substructures within thesedomains that actually come in contact with the DNA fall into one ofa rather small group of recognizable and characteristic structuralmotifs.Before examining these protein structures, it is useful to considerthe recognition surfaces on the DNA with which regulatory proteinsmust interact.
Most of the groups that differ from one base to anotherand can therefore permit discrimination between base pairs are hydrogen-bond donor and acceptor groups exposed in the major DNAgroove (Fig. 27-9). Most of the protein-DNA contacts that impartspecificity are therefore hydrogen bonds. One notable exception is anonpolar surface near C-5 of pyrimidines, where thymine is readilydistinguished from cytosine by virtue of thymine's protruding methylgroup (Fig. 27-9).
Protein-DNA contacts are also possible in the minorgroove of the DNA, but the hydrogen-bonding patterns here generallydo not allow ready discrimination between different base pairs.Major grooveMajor grooveMajor grooveMinor grooveMinor grooveHMinor groovemsmmMinor grooveChapter 27 Regulation of Gene ExpressionH OH 0I IIR-N-C-C-R'Glutamine(or asparagine)R-N-C-C-R'ArginineHJL—HCH 3Thymine: AdenineCytosine: GuanineAs for the regulatory proteins themselves, the amino acid residueswhose side chains are most often found hydrogen-bonded to bases inthe DNA include Asn, Gin, Glu, Lys, and Arg. Is there a simple "recognition code" in which an amino acid is always paired with a certainbase? The two hydrogen bonds that can form between Gin or Asn andthe N 6 and N-7 positions of adenine (Fig.
27-10) constitute a patternthat cannot form with any other base. An Arg residue can similarlyform two hydrogen bonds to both N-7 and O6 of guanine (Fig. 27-10).However, examination of the structures of many DNA-binding proteins has shown that there are multiple ways for a protein to recognizeeach base pair, and no simple code exists. The Gln-adenine interactionspecifies A=T base pairs in some cases, whereas a van der Waalspocket for the methyl group of thymine is the mechanism used to recognize A=T base pairs in other proteins. It is not yet possible to examinethe structure of a DNA-binding protein and infer the sequence of theDNA to which it binds.The DNA-binding domains of regulatory proteins tend to be small(60 to 90 amino acid residues).
Only a small subset of the amino acidswithin these domains actually contact the DNA, and the structure ofthe protein in the region where these amino acids occur is not random.Two structural motifs that play a major role in DNA binding have beenfound in numerous regulatory proteins: the helix-turn-helix motifand the zinc finger.
Other DNA-binding motifs exist in some proteins,but the discussion here focuses on these well-studied examples.The Helix-Turn-Helix This DNA-binding motif was the first to bestudied in detail. It is the physical basis for protein-DNA interactionsfor many prokaryotic regulatory proteins. Closely related DNA-binding motifs also occur in some eukaryotic regulatory proteins. The helixturn-helix motif consists of two short a-helical segments 7 to 9 aminoacid residues long, separated by a /3 turn (about 20 amino acids total).This structure generally is not stable by itself, but it represents thereactive portion of the larger DNA-binding domain. One of the two ahelices is referred to as the recognition helix, because it usually contains many of the amino acids that interact with the DNA; this helix ispositioned in the major groove. The Cro repressor protein from bacteriophage 434 (a close relative of bacteriophage A) provides a good examDie (Fie.
27-11).949Figure 27-10 Two examples of specific aminoacid—base pair interactions that have been observedin the structures of DNA-bound regulatory proteins.950Part IV Information Pathwaysi4(b)•;«#••#11Figure 27—11 The Cro repressor of bacteriophage434 and its interaction with DNA. Each subunit ofthis dimeric protein contains 71 amino acids. It ispresented as a ribbon in (a) and (b), alone andcomplexed with its specific DNA binding site.
Thetwo subunits are shown in gray and light blue, except for the helix-turn-helix motif in each which isshown in red and yellow. The red helices are therecognition helices, which are positioned in adjacentmajor grooves of the DNA as seen in (b). The interactions between protein and DNA that allow thisrepressor to discriminate between its specific DNAbinding site (shown here) and other DNA sequencesare illustrated in (c) and (d). The protein subunitsare again shown in gray and light blue; chemicalgroups on both the DNA and protein that interactthrough hydrogen bonds or van der Waals (hydrophobic) interactions are highlighted in red and orange, respectively.
Discrimination is mediated by(d)interactions between each protein subunit and fourbases (the DNA binding site is a palindrome, andthe interactions are the same for both subunits).One set of hydrogen bonds is formed between a Ginresidue and the N6 and N-7 of an adenine (see Fig.27—10); another hydrogen bond is formed betweenthe O6 of a guanine and another Gin.
In addition,van der Waals pockets on each subunit bind to theC-5 methyl groups of two adjacent thymines. Thecomplementary interacting groups are evident in(c), and the complex is shown in (d). Many nonspecific contacts (not shown) also exist between proteinand DNA in this complex. These do not contributeto discrimination between DNA sequences, but docontribute to the overall DNA-binding affinity. Aninteresting feature of this structure is that theDNA is bent slightly when it is bound.
This occursin the binding of many proteins to DNA (see Fig.27-16.)Chapter 27 Regulation of Gene ExpressionT/ie Zinc Finger Zinc fingers consist of about 30 amino acid residues;four of the residues, either four Cys or two Cys and two His, coordinatea single Zn 2+ atom (Fig. 27-12). This structural motif is found in manyeukaryotic DNA-binding proteins, with several often present in a single protein. There are few, if any, known examples among prokaryoticproteins. Bacteriophage T4 has a protein, the gene 32 protein, thatbinds single-stranded DNA. It binds a single zinc atom within a structure that may be similar to a zinc finger.
An apparent record is held bya DNA-binding protein derived from the frog Xenopus, which has 37zinc fingers. The precise manner in which proteins containing zinc fingers bind to DNA may vary from one protein to the next. In some casesthese structures contain the amino acid residues that are involved insequence discrimination; in other cases the zinc fingers appear to bindDNA nonspecifically, and the amino acids required for specificity arefound elsewhere in the protein.
The interaction of three zinc fingers(derived from a mouse regulatory protein called Zif 268) with DNA isshown in Figure 27-12b. It should be noted that some regulatory proteins contain zinc bound within structures that are distinct from thezinc finger.Regulatory Proteins Also Interact with Other ProteinsRegulatory proteins generally contain additional domains that are involved in interactions with RNA polymerase, other regulatory proteins, or additional copies of the same regulatory protein (Fig. 27-13).The DNA binding sites for regulatory proteins are generally invertedrepeats of a short DNA sequence (a palindrome) at which two or fourcopies of a regulatory protein bind cooperatively, as in Figures 27-11and 27-13.The Lac repressor is a tetramer of identical subunits (Mr 37,000).
Awild-type E. coli cell generally contains about ten copies of Lac repressor. The i gene is transcribed from its own promoter independently ofthe lac operon genes (Fig. 27-7). The repressor binds to a palindromicoperator sequence that spans 22 base pairs within the larger regula-951Figure 27-12 Zinc fingers, (a) A ribbon representation of a single zinc finger derived from the regulatory protein Zif 268.
The zinc atom is in orangeand the amino acid residues that coordinate it (twoHis and two Cys) are shown in red. (b) Three zincfingers (light blue and gray) from Zif 268 areshown complexed with DNA. The zinc atoms areagain shown in orange.1111Figure 27-13 The bacteriophage A repressorbound to DNA. The two identical subunits of thedimeric protein are shown in gray and light blue.RNApolymerase(a)araO2DNACAP binding sitearalaraBADaraCara OirBADaraC mRNAaraOo(b)araCaraOxAraC'proteinsBADbindingsite(c)RNApolymerasearaCaraBADaraO2Figure 27-20 Regulation of the ara operon.(a) When AraC protein is depleted, the araC geneis transcribed from its own promoter, (b) Whenarabinose levels are low and glucose levels high,AraC protein binds to both aral and araO2 andbrings these sites together to form a DNA loop.The operon is repressed in this state.
AraC proteinalso binds to araOi, repressing further synthesis ofAraC. (c) When arabinose is present and glucoseconcentration is low, AraC protein binds arabinoseand changes conformation to become an activator.The DNA loop is opened, and the AraC proteinacts in concert with CAP-cAMP to facilitatetranscription.aralCAP•bindingsiteArabinoseBAD/\/\/\/\/varaBAD mRNAUnder these conditions, the AraC protein bound to araO2 and thatbound to aral bind to each other, forming a DNA loop of about 210 basepairs.
In this configuration the system represses transcription fromthe promoter for the araBAD genes (Fig. 27-20b). (2) Glucose is notpresent (or is at low levels) but arabinose is available. Under theseconditions, CAP-cAMP becomes abundant and binds to its site adjacent to aral. Arabinose also binds to the AraC protein, altering itsconformation. The DNA loop is opened, and the AraC protein bound ataral now becomes an activator, acting in concert with CAP-cAMP toinduce transcription of the araBAD genes (Fig. 27-20c). (3) Arabinoseand glucose are both abundant. (4) Arabinose and glucose are bothabsent. For both (3) and (4), the status of the system is not entirelyclear, but it remains repressed in both cases. The ara operon is a complex regulatory system that provides rapid and reversible responses tochanges in environmental conditions.Genes for Amino Acid Biosynthesis AreRegulated by Transcription AttenuationAmino acids are required in large amounts for protein synthesis, andE.
coli has enzymes for synthesizing all of them. Not surprisingly, thegenes for the enzymes needed to synthesize a given amino acid aregenerally clustered in an operon. These enzymes are needed, andhence the operon corresponding to an amino acid is expressed, whenever existing supplies of the amino acid are inadequate for cellularrequirements. When the amino acid is in abundant supply, the biosynthetic enzvmes are no longer needed and the oneron is renressed.Chapter 27 Regulation of Gene ExpressionTrpTrprepressor959Figure 27-21 The trp operon and tryptophan biosynthesis.
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