生物化學(xué)原理課件（英文）：Chapter42 Prokaryotic regulation of gene expression

1、Chapter42 Prokaryotic regulation of gene expression Outline Negative vs positive regulation Regulation at the DNA level Regulation at the transcription level 1. Regulation of Transcription Initiation: Operons( inducible/repressible) 2. Regulation at Steps after Transcription Initiation- Attenuation

2、and Antitermination Regulation at the translation level Regulation induced by environmental siganls Global regulation at the genome level Negative vs Positive Regulation Negative regulationProtein (repressor) inhibits transcription (Ex. LexA). Inducer binds to repressor, alters form, reduces affinit

3、y for target, allows expression of gene. Sometimes, small molecule required for repressor activity. Positive regulationActivator protein increases transcription rate. Generally bound to a smaller signal molecule. THE PROBLEM Prokaryotes have to monitor its environment and accordingly change its meta

4、bolism Monitor pH, source of energy, galactose, amino acid residues, O2 Prokaryotes must accomplish specialized functions in one unspecialized cell Options Have all gene products functioning at all times (constitutive expression) Turn on genes only as they are needed (inducible expression) Gene expr

5、ession is controlled by regulatory proteins In bacteria , signals are communicated to genes by regulatory proteins, which come in two types: activators and repressors. Many promoters are regulated by activators that help RNA polymerase bind DNA and by repressors that block that binding In the absenc

6、e of both activator and repressor , RNA polymerase occasionally binds the promoter spontaneously and initiates a low level of transcription. The repressor blocks polymerase binding to the promoter. The site on DNA where a repressor binds is called an operator. An activator helps polymerase bind the

7、promoter. this mechanism, often called recruitment, is an example of cooperative binding of proteins to DNA. Not all promoters are limited in the same way. In some cases, closed complex does not spontaneously undergo transition to the open complex, so an activator must stimulate the transition from

8、closed to open complex. Activators interact with the stable closed complex and induce a conformational change that causes transition to the open complex. This mechanism is an example of allostery. Effect of activator and repressor on transcription Some activators work by allostery and regulate steps

9、 after RNA polymerase binding Action at a distance and DNA looping a.Cooperative binding of proteins to adjacent sites. b.Cooperative binding of proteins to separated sites. DNA-bending protein can facilitate interaction between DNA-bending proteins Sigma factor cascade Alternative factors direct RN

10、A polymerase to alternative sets of promoters. one of these alternatives is the heat shock factor, 32; another example of an alternativefactor, 54. The phage SPO1 uses three factors in the succession to regulate expression of its genome The lac Operon LactoseA disccharide hydrolyzed to glucose and g

11、alactose . Lactose metabolizing enzymes expresse as a polycistronic message ( lacZ, lacY, lacA). Is an inducible operon. Consists of Regulatory components Structural components The Players ERegulatory Promoter (P) Operator (O) LacI EStructural lacZ lacY lacA An activator and a repressor together con

12、trol the Lac genes E These genes are expressed at high levels only when lactose is available, and glucose- the preferred energy source-is not. E Two regulatory proteins are involved: one is an activator called CAP, the other a repressor called the Lac repressor. E Each of these respond environmental

13、 signal and communicates it to the Lac genes. Lactose metabolism The three Lac genes-LacZ, LacY, LacA-are arranged adjacently on the E.coli genome and are called the Lac operon. They are transcribed as a single m RNA from the promoter. Expression of the Lac genes The lac operon model Lac promoter se

14、quence CAP vs Lac Repressor CAP and Lac repressor have opposing effects on RNA polymerase binding to the Lac promoter The presence or absence of the sugars lactose and glucose control the level of the Lac genes. High levels of expression require the presence of lactose and absence of the preferred e

15、nergy source, glucose. CAP and Lac repressor are shown as single units, but CAP actually binds DNA as a dimer, and Lac repressor binds as a tetramer. CAP recruits polymerase to the Lac promoter where it spontaneously undergoes isomerization to the open complex. The Lac operator overlaps the promoter

16、, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter and thus initiating RNA synthesis. CAP binds as a dimer to a site similar in length to the Lac operator, but different in sequence. When CAP binds to that site, the activator helps polymerase bin

17、d to the promoter by interacting with the enzyme and recruiting it to the promoter. The site bound by Lac repressor is called the Lac operator. The symmetric half-sites of the Lac operator. The colored bars above and below the DNA show regions covered by RNA polymerase and the regulatory proteins Th

18、e control region of the Lac operon CAP has separate activating and DNA-binding domains CAP activates the Lac genes by simple recruitment of RNA polymerase. Mutant versions of CAP have been isolated that bind DNA but do not activate transcription. (positive control) The amino acid substitutions in th

19、e positive control mutants identify the region of CAP that touches polymerase, called the activating region. RNA polymerase binding at the Lac promoter with the help of CAP. CAP is recognized by the CTDs of the CTDs of the subunits. Activation of the Lac promoter by CAP CAP and Lac repressor bind DN

20、A using a common structure motif Although the details of DNA binding for bacterial activators and repressors differ (including CAP and the Lac repressor), the basic mechanism of DNA recognition is similar for most bacterial regulators. In the typical case, the protein binds as a homodimer to a site

21、that is an inverted repeat. One monomer binds each half-site, with the axis of symmetry of the dimer lying over that of the binding site. Recognition of specific DNA sequences is achieved using a conserved region of secondary structure called a helix-turn helix. This domain is composed of two helice

22、s, one of which- the recognition helix. Lac repressor binds as a tetramer to two operators. Each operator is contacted by only two of these subunits. The active of Lac repressor and CAP are controlled allosterically by their signals The conversion of lactose to allolactose is catalyzed by - galactos

23、idase, itself encoded by one of the Lac genes. Allolacrose binds to Lac repressor and triggers a change in the shape of that protein. CAP activity is regulated in a similar manner. Only when glucose levels are low does CAP bind DNA and activate the Lac genes. Then c AMP is separate from the part of

24、the protein that binds DNA. lac operon: summary Subject to both positive and negative regulation Negatively regulated (repressed) by lac repressor under conditions of lactose deprivation. Positively regulated (activated) by CAP/CRP under conditions of glucose deprivation. Together, these two mechani

25、sms result in a 1 300-fold range of expression. Ara Operon Dual action regulatory protein- AraC (-) arabinose:Represses (+) arabinose:Activates AraI :AraI1 ;AraI2 Operators AraO1- regulates AraC AraO2- regulates AraBAD CAPcAMP binding site. Increases transcription. Autoregulation of AraC AraC transc

26、ribed from Pc. Pc regulated by O1. As level of AraC rises, binds to AraO1 and prevents transcription from Pc. prevents wasteful accumulation of repressor Is an example of autoregulation AraC and control of the araBAD operon by antiactivation Control of the araBAD operon. The promoter of the araBAD o

27、peron from E.coli is activated in the presence of arabinose and the absence of glycose and directs expression of genes encoding enzymes required for arabinose metabolism.two activators work together here: AraC and CAP. Exhibit both negative and positive control The araC and araBAD operons Mechanism

28、of araBAD regulation Repression at a distance mediated by DNA looping araBAD operon: summary Subject to both positive and negative regulation: 1.Negatively regulated by araC under conditions of arabinose deprivation. 2. Positively regulated by CAP/CRP under conditions of glucose deprivation. 3.Posit

29、ively regulated by araC under conditions of arabinose abundance. Trp Operon nEncodes enzymes necessary for Trp synthesis encodes a set of anabolic enzymes rather than catabolic enzymes. Anabolic enzymes are generally turned off by presence of a product (feedback inhibition) nIn addition to repressio

30、n, system shows attenuation, a finer level of control. nStructure 5 structural genes3 enzymes Promoter and operator precede structural genes In absence of Trp, TrpR protein is inactive nLow tryptophan, No repression,transcription activated nHigh tryptophan, Tryptophan (a corepressor) combines with f

31、ree repressor dimer (aporepressor dimer)=repressor dimer, transcription blocked The E. coli trp operon Amino acid biosynthetic operons are controlled by premature transcription termination (attenuation) In E.coli the five contiguous trp genes encode enzymes that synthesize the amino acid tryptophan.

32、 Transcription termination at the trp attenuator When the tryptophan concentration is low, the Trp repressor is free of its corepressor and vacates its operator, allowing the synthesis of trp mRNA to commence from the adjacent promoter. There is a second hairpin that can form between region1 and 2 o

33、f the leader Region 2 also is complementary to region 3; thus, yet another hairpin consisting of region2 and 3 can form, and when it does it prevents the terminator hairpin from forming The leader RNA contains an open-reading frame encoding a short leader peptide of 147 amino acids, and this open- r

34、eading frame is preceded by a strong ribosome binding site Trp operator leader RNA Transcriptional attenuation of the trp operon is regulated by abundance of charged tRNATrp mRNA mRNA * Ribosome translating Terminator and anti-terminator conformations of the trp leader (trpL) sequence Terminator (At

35、tenuator): GC-rich stem followed by a series of U residues 2-3 Anti-Terminator: Prevents formation of 3-4 stem loop trp operon: summary Two negative regulatory mechanisms 1.trp repressor binding to trpO operator, thereby sterically occluding the trp promoter (trpP). 2.ribosome facilitating formation

36、 of the terminator (attenuator) stem-loop structure within the 5-end of the trp mRNA(by occupying segment 2 within trpL). contribute to the 700-fold dynamic range of transcription. Regulation of Other Amino Acid Biosynthesis Operons EAttenuation is involved in regulating many operons. EWhen the oper

37、on is for amino acid biosynthesis, the leader sequence always includes codons for that amino acid. EOther operons regulated by attenuation include rRNA (rrn) and E. coli ampicillin resistance (ampC). Predicted amino acid sequences of the leader peptides of a number of attenuator-controlled bacterial

38、 operons Environmentally-responsive adaptation External stimuli Internal stimuli Membrane- permeable signals Transmembrane receptor Indirect effect Physiological change Gene Regulation The two-component system 30 such systems in E. coli also found in plants and fungi 1.Sensor histidine kinase (HK) m

39、ay or may not be transmembrane phosphorylates itself 2.Response regulator (RR) often, but not always affects gene expression phosphorylated by HK Basic model for a two component-regulatory system Autoregulation at the translational level Ribosomal proteins are translational repressors of their own s

40、ynthesis Regulation of translation often works in a manner analogous to transcriptional repression: a “repressor” binds to the translation start site and blocks initiation of that process. Control of ribosomal protein genes is simplified by their organization into several operons, each containing ge

41、nes for up to 11 ribosomal proteins. When extra copies of a ribosomal protein operon are introduced into the cell, the amount of mRNA increases. The cell compensates for extra mRNA by curtailing its activity as a template. For each operon, one( or a complex of two) ribosomal proteins binds the messe

42、nger near the translation initiation sequence of one of the first genes in the operon, preventing ribosomes from binding and initiating translation. E.coli ribosomal protein operons Ribosomal protein S8 binds 16S rRNA nHow one protein can function both as a ribosomal component and as a regulator of

43、its own translation is shown by comparing the sites where that protein binds to rRNA and to its mRNA. Riboswitches RNA structure is more flexible RNA directly interacts with metabolites + no need for sensory proteins Regulation of syntheses of thiamine pyrrophosphate 5end of sequence conserved seque

44、nce box binds TPP Binding TPP switch in conformation resulting in shielding SD sequence Metabolite directly regulates mRNA translation other riboswitches: syntheses of coenzyme B12, riboflavin and S-adenosyl methionine Regulation of Gene Expression by Riboswitches TPP riboswitch Riboswitch Control o

45、f the Riboflavin Operon of Bacillus subtilis Regulation of Translation by a Riboswitch The CRISPR Antiviral Defense System CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The CRISPR region on the bacterial chromosome is essentially a memory bank of hostile virus sequence

46、s. It consists of many different segments of virus sequence alternating with identical repeated sequences. The CRISPR system provides resistance to any viruses that contain the same or very closely related sequences. The proteins of the CRISPR system ( CAS proteins) perform two roles. Some use the s

47、tored sequence information to recognize intruding virus genomes and destroy them. Others are involved in obtaining and storing segments of virus sequence, a process that remains obscure. The CAS proteins are encoded by genes that lie upstream of the CRISPR sequences. The CRISPR region is transcribed

48、 as a whole into a long RNA molecule that is then cleaved by CAS proteins in the middle of each of the repeated sequences. This converts it into individual virus-specific segments. If one of these segments base-pairs with the nucleic acid of an invading virus, then the virus DNA or RNA is destroyed

49、by other CAS proteins. The CRISPR system is widely distributed in both Archaea and Bacteria. Approximately 90% of the sequenced genomes of Archaea and 70% of those of Bacteria possess the CRISPR system. Operation of the CRISPR system The case of phage - layers of regulation Phage use many bacterial

50、components for replication, and control their use with phage gene products. Bacteriophage has two possible pathways when it enters its E. coli host: 1.The lytic cycle, in which the phage takes over the cell and produces progeny phage. 2.The lysogenic cycle, where phage chromosome is inserted into th

51、e E. coli chromosome, and replicates with the bacterial genome. The life-cycle of phage Early Transcription Events The chromosome is linear, with “sticky” ends used to circularize it in the host cell. The regulatory system for choosing between the lytic and lysogenic pathways is contained in the chr

52、omosome. a. First, transcription begins at promoters PL (leftward transcription) and PR (rightward) i. The first gene transcribed from PR is cro (control of repressor and other). The Cro protein is involved in the genetic switch to the lytic pathway. ii. The first protein transcribed from PL is N, w

53、hich is a transcription antiterminator that allows RNA synthesis to go through termination regions into the early genes. (1)N protein allows expression of the cII protein, which in turn activates: (a)cI ( repressor) (b)O and P (DNA replication proteins). (c)Q (activation of late genes for lysis and

54、phage particle proteins, only when Q protein accumulates to certain levels). Expression of genes after infecting E. coli and the transcriptional events that occur when either the lysogenic or lytic pathways are followed The Lysogenic Pathway 1.Early transcription events determine whether the lytic or lysogenic pathway occurs. 2.The lysogenic pathway results when cII and cIII are expressed. a.The action of cII and cIII proteins activates the PRE promoter, causing transcription of the cI ( repressor) gene. b. repressor binds to 2 operator regions, OL and OR, whose sequen