页数：70页文件大小：1.34 MB来源： 分子生物学基因Geneppt课件
本课件为《分子生物学》第10章操纵子（The operon），依据 Gene VII 教材编排，沿Structural gene、Repressor等小节推进，讲解本章核心概念与分子机制，配逐页图示与文字版。
Chapter 10 The operon
Figure 10.1 A regulator gene codes for a protein that acts at a target site on DNA.
Figure 10.2 In negative control, a trans-acting repressor binds to the cis-acting operator to turn off transcription. In prokaryotes, multiple genes are controlled coordinately.
Figure 10.3 In positive control, trans-acting factors must bind to cis-acting sites in order for RNA polymerase to initiate transcription at the promoter. In a eukaryotic system, a structural gene is controlled individually.
Operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in DNA recognized by regulator gene product(s).
Figure 10.4 The lac operon occupies ~6000 bp of DNA. At the left the lacI gene has its own promoter and terminator. The end of the lacI region is adjacent to the promoter, P. The operator, O, occupies the first 26 bp of the long lac Zgene, followed by the lac Yand lac Agenes and a terminator.
Figure 10.5 Repressor and RNA polymerase bind at sites that overlap around the startpoint of the lac operon.
Gratuitous inducers resemble authentic inducers of transcription but are not substrates for the induced enzymes.
Figure 10.6 Addition of inducer results in rapid induction of lac mRNA, and is followed after a short lag by synthesis of the enzymes; removal of inducer is followed by rapid cess at ion of synthesis.
Figure 10.7 Repressor maintains the lac operon in the inactive condition by binding to the operator; addition of inducer releases the repressor, and thereby allows RNA polymerase to initiate transcription.
Figure 10.8 Operator mutations are constitutive because the operator is unable to bind repressor protein; this allows RNA polymerase to have unrestrained access to the promoter. The Ocmut at ions are cis-acting, because they affect only the contiguous set of structural genes.
Figure 10.9 Mutations that inactivate the lac Igene cause the operon to be constitutively expressed, because the mutant repressor protein cannot bind to the operator.
Figure 10.10 Mutations map the regions of the laclgene responsible for different functions. The DNA-binding domain is identified by lacI-dmut at ions at the N-terminal region; lacl-mut at ions unable to form tetramers are located between residues 220-280. Other lacI-mut at ions occur throughout the gene. lac Ismut at ions occur in regularly spaced clusters between residues 62-300.
Figure 10.11 The lac operator has a symmetrical sequence. The sequence is num be red relative to the startpoint for transcription at +1. The regions of dyad symmetry are indicated by the shaded blocks.
Figure 9.16 One face of the promoter contains the contact points for RNA.
Figure 10.12 Does the inducer bind to free repressor to upset an equilibrium (left) or directly to repressor bound at the operator (right)?.
Figure 10.13 The structure of a monomer of Lac repressor identifies several independent domains. Photograph kindly provided by Mitchell Lewis.
Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.
Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.
说明：本页源文与上一页完全相同，疑为课件连续两页沿用同一页内容，已按原文保留。
Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.
说明：本页源文与上一页完全相同，疑为课件连续两页沿用同一页内容，已按原文保留。
Figure 10.14 The crystal structure of the core region of Lac repressor identifies the interactions between monomers in the tetramer. Each monomer is identified by a different color. Photographs kindly provided by Alan Friedman.
说明：本页源文与上一页完全相同，疑为课件连续两页沿用同一页内容，已按原文保留。
Figure 10.15 Inducer changes the structure of the core so that the headpieces of a repressor dimer are no longer in an orientation that permits binding to DNA. Photographs kindly provided by Mitchell Lewis.
Figure 10.16 When a repressor tetramer binds to two operators, the stretch of DNA between them is forced into a tight loop. (The blue structure in the center of the looped DNA represents CAP, another regulator protein that binds in this region). Photograph kindly provided by Mitchell Lewis.
Figure 10.17 Lac repressor binds strongly and specifically to its operator, but is released by inducer. All equilibrium constants are in M-1.
Figure 10.18 Virtually all the repressor in the cell is bound to DNA.
Figure 9.12 How does RNA polymerase find target promoters so rapidly on DNA?.
Autogenous control describes the action of a gene product that either inhibits (negative autogenous control) or activates (positive autogenous control) expression of the gene coding for it.
Figure 10.19 The trp repressor recognizes operators at three loci. Conserved bases are shown in red. The location of the mRNA varies, as indicated by the red arrows.
Figure 10.20 Operators may lie at various positions relative to the promoter.
Derepressedstate describes a gene that is turned on. It is synonymous with induced when describing the normal state of a gene; it has the same meaning as constitutive in describing the effect of mutation.
Figure 10.2 In negative control, a trans-acting repressor binds to the cis-acting operator to turn off transcription. In prokaryotes, multiple genes are controlled coordinately.
Figure 10.3 In positive control, trans-acting factors must bind to cis-acting sites in order for RNA polymerase to initiate transcription at the promoter. In a eukaryotic system, a structural gene is controlled individually.
Figure 10.21 Control circuits are versatile and can be designed to allow positive or negative control of induction or repression.
Catabolite repression describes the decreased expression of many bacterial operons that results from addition of glucose. It is caused by a decrease in the level of cyclic AMP, which in turn inactivates the CAP regulator.
Figure 10.22 Cyclic AMP has a single phosphate group connected to both the 3′and 5′positions of the sugar ring.
Figure 10.21 Control circuits are versatile and can be designed to allow positive or negative control of induction or repression.
Figure 10.23 Glucose causes catabolite repression by reducing the level of cyclic AMP.
Figure 10.24 The consensus sequence for CAP contains the well conserved pent a mer TGTGA and (sometimes) an inversi on of this sequence (TCANA).
Figure 10.25 The CAP protein can bind at different sites relative to RNA polymerase.
Figure 10.26 Gel electrophoresis can be used to analyze bending.
Figure 10.27 CAP bends DNA >90°around the center of symmetry.
Figure 10.28 Stringent factor catalyzes the synthesis of ppp Gpp and pp Gpp; ribosomal proteins can dephosphorylate ppp Gpp to pp Gpp.
Figure 10.29 In normal protein synthesis, the presence of aminoacyl-tRNA in the A site is a signal for peptidyl transferase to transfer the polypeptide chain, followed by movement catalyzed by EF-G; but under stringent conditions, the presence of uncharged tRNA causes RelA protein to synthesize (p)pp Gpp and to expel the tRNA.
Figure 10.30 Nucleotide levels control initiation of rRNA transcription.
Figure 10.35 Translation of the r-protein operons is autogenously controlled and responds to the level of rRNA.
Figure 10.31 Aregul at or protein may block translation bybinding to a site onmRNA that overlaps the ribosome-binding site at the initiation codon.
Figure 10.32 Proteins that bind to sequences within the initiation regions of mRNAs may function as translational repressors.
Figure 10.33 Secondary structure can control initiation. Only one initiation site is available in the RNA phage, but translation of the first cistron changes the conform at ion of the RNA so that other initiation site(s) become available.
Figure 10.34 Genes for ribosomal proteins, protein synthesis factors, and RNA polymerase subunits are interspersed in a small number of operons that are autonomously regulated. The regulator is named in red; the proteins that are regulated are shaded in pink.
Figure 10.35 Translation of the r-protein operons is autogenously controlled and responds to the level of rRNA.
Figure 10.36 Excess gene 32 protein (p32) binds to its own mRNA to prevent ribosomes from initiating translation.
Figure 10.37 Gene 32 protein binds to various substrates with different affinities, in the order single-stranded DNA, its own mRNA, and other mRNAs. Binding to its own mRNA prevents the level of p32 from rising >10-6M.
Figure 10.38 Tubulin is assembled into microtubules when it is synthesized. Accumul at ion of excess free tubulin induces instability in the tubulin mRNA by acting at a site at the start of the reading frame in mRNA or at the corresponding position in the nascent protein.
Figure 10.39 Attenuation occurs when a terminator hairpin in RNA is prevented from forming.
Figure 10.40 Termination can be controlled via changes in RNA secondary structure that are determined by ribosome movement.
Figure 10.41 The trp operon consists of five contiguous structural genes preceded by a control region that includes a promoter, operator, leader peptide coding region, and attenuator.
Figure 10.42 An attenuator controls the progression of RNA polymerase into the trp genes. RNA polymerase initiates at the promoter and then proceeds to position 90, where it pauses before proceeding to the attenuator at position 140. In the absence of tryptophan, the polymerase continues into the structural genes (trp Estarts at +163). In the presence of tryptophan there is ~90% probability of termination to release the 140-base leader RNA.
Figure 10.43 The trp leader region can exist in alternative base-paired conform at ions. The center shows the four regions that can base pair. Region 1 is complementary to region 2, which is complementary to region 3, which is complementary to region 4. On the left is the conform at ion produced when region 1 pairs with region 2, and region 3 pairs with region 4. On the right is the conform at ion when region 2 pairs with region 3, leaving regions 1 and 4 unpaired.
Figure 10.44 The alternatives for RNA polymerase at the attenuator depend on the location of the ribosome, which determines whether regions 3 and 4 can pair to form the terminator hairpin.
Figure 10.45 Antisense RNA can affect function or stability of an RNA target.
Figure 10.46 Increase in osmolarity activates EnvZ, which activates OmpR, which induces transcription of mic Fand ompC(not shown). micF RNA is complementary to the 5′region of omp FmRNA and prevents its translation.
Figure 10.47 lin4 RNA regulates expression of lin14 by binding to the 3′nontranslated region.
Figure 10.48 Antisense RNA can be generated by reversing the orientation of a gene with respect to its promoter, and can anneal with the wild-type transcript to form duplex RNA.
1. Transcription is regulated by the interaction between trans-acting factors and cis-acting sites. 2. Initiation of transcription is regulated by interactions that occur in the vicinity of the promoter. 3. A repressor protein prevents RNA polymerase either from binding to the promoter or from activating transcription. 4. The ability of the repressor protein to bind to its operator is regulated by a small molecule.
Summary
5. The lactose pathway operates by induction, when an inducer -galac to side prevents the repressor from binding its operator; transcription and translation of the lacZ gene then produce -galactosidase, the enzyme that metabolizes -galac to sides.6. Some promoters cannot be recognized by RNA polymerase (or are recognized only poorly) unless a specific activator protein is present. 7. A protein with a high affinity for a particular target sequence in DNA has a lower affinity for all DNA. 8. Gene expression can be controlled at stages subsequent to transcription. 9. The level of protein synthesis itself provides an important coordinating signal.
Summary
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