21NEWGene Regulation in Bacteria and Bacteriophages

21NEWGene Regulation in Bacteria and Bacteriophages - t...

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Unformatted text preview: t alking about protein when we're talking about trans mediated by genes that encode regulatory proteins cis - a regulatory element of DNA that has to do whether the gene is being t urned on or turned offmediated by DNA s equences The interactions between regulatory proteins and DNA sequences illustrated in this experiment have led to the definition of two genetic terms. A trans-effect is a form of genetic regulation that can occur even though two DNA segments are not physically adjacent. The action of the lac repressor on the lac operon is a trans-effect. In contrast, a cis-acting element is a DNA segment that must be adjacent to the gene(s) that it regulates; it is said to have a cis-effect on gene expression. The lac operator site is an example of a cis-acting element. A trans-effect is mediated by genes that encode regulatory proteins; a cis-effect is mediated by DNA sequences that are bound by regulatory proteins. if glucose is around, bacterium will preferentially use glucose even if lactose is there too. repressor is trans element - it is a protein glucose is low, cyclic amp is high if glucose is high, c yclic amp is low allolactose is an effector and is an inducer because it t urns it on from off balancing act of a positive system and a negative system The lac operon is also regulated by an activator protein. A second mechanism of lac operon regulation is called catabolite repression. The presence of glucose in the cell represses the lac operon. Glucose is a catabolite of cellular metabolism. If placed in an environment containing both glucose and lactose, E. coli will use the glucose first. This sequential use of sugars is called diauxic growth. The effector molecule for this pathway is cyclic AMP (cAMP). This molecule is produced from ATP by adenyl cyclase. cAMP binds to an activator protein called CAP (catabolite activator protein). This is an inducible system, under positive control (Figure 14.8). The binding of cAMP to CAP causes the cAMP-CAP complex to bind to the CAP site near the lac promoter. This increases the rate of transcription. Further studies have revealed that the lac operon has three operator sites for the lac repressor. The study of mutations in the lac operon has led to the identification of three operator sites in the lac operon (Figure 14.9). It is believed that the repressor must bind to two of the three operator sites to repress transcription. One CAP site sites must be O1 (Figure O3 is located upstream from the of these and the lac promotoer (P) O3 ..CAP site....P.... O1...lac Z.... O2... binding of !repressor to the two creates looping that prevents t ranscription 14.9). no glucose, no lactose ---> attaches to O1 and O3 remember: promoter has -10 and -35, RNA pol attaches and sigma factor fi nds the -35 and -10 sites can't get RNA pol on there "! Each repressor dimer binds to one operator site, so the repressor tetramer brings the two operator sites together. This causes the formation of a DNA loop in the intervening region (Figure 14.10). c atabolic high glucose, low c yclic amp, no c omplex on cap s ite, no GO if glucose is around, you want t his thing OFF The ara operon can be regulated positively or negatively by the same regulatory protein. The ara operon in E. coli is involved with arabinose (sugar) metabolism. The organization of the ara operon is illustrated in Figure 14.11. The AraC protein can act as a repressor or activator of transcription, depending on the presence of arabinose. In the absence of arabinose, AraC acts as a repressor (Figure14.12a). trp is anabolic we want it to be t urned OFF when t ryptophan is high, we want it ON when tryptophan is low tryptophan acts as a c orepressor to repress In the presence of arabinose, AraC acts as an activator (Figure 14.12b). The trp operon is regulated by a repressor protein and also by attenuation. The trp operon encodes enzymes that are needed for the biosynthesis of the amino acid tryptophan (Figure 14.13). trpL and trpR are involved in regulation. trpE, trpD, trpC, trpB and trpA encode enzymes for biosynthesis. trpR encodes the trp repressor. When tryptophan levels are low, the trp repressor does not bind to the operator site, and transcription of the operon proceeds. When tryptophan is present, it acts as a corepressor, binding to the trp repressor and allowing it to bind to the operator. This inhibits transcription. trpL encodes a short peptide called the Leader peptide, which functions in attenuation. A second mechanism of regulation, called attenuation (Figure 14.13c), was discovered by Yanofsky (1970s) by studying mutant strains that lacked the trp repressor. Attenuation occurs in bacteria due to the coupling of transcription and translation. During attenuation, transcription begins, but is not finished. An area of DNA called the attenuator regulates this process and stops transcription of the tryptophan biosynthesis enzymes. The mechanism of attenuation is presented in Figures 14.14 and 14.15. Several metabolic pathways for amino acid production are regulation through attenuation. if trp is high 3-4 stem loop and the last piece of RNA will force the RNA pol off the promoter if low, no corepressor to attach to operator...get 2-3 stem loop, double GO type of signal 3 and 4 makes the whole thing stop, and makes t ranscription stop if trp is low... if no t rp, it cannot hydrogen bond to number two region, t hen number 2 makes a loop with 3.... 2-3 loop is a GO signal...now we're gonna make t rp becuase now 3 and 4 are not ! bound and going to t ranscribe the rest of the genes now #! c atabolic - lac operon and lactose anabolic - trp operon and t rpytophan Inducible operons usually encode catabolic enzymes and repressible operons usually encode anabolic enzymes. Catabolic genes in an operon are usually regulated in an inducible manner, with the substrate usually acting as the inducer. Anabolic genes in an operon are usually regulated in a repressible manner, with the corepressor (and sometimes the inhibitor) being the molecule produced by the operon. Translational and Posttranslational Regulation The majority of gene regulation in bacteria is at the transcriptional level. Some regulation occurs during initiation, elongation, and termination of translation. Posttranslational regulation refers to the functional control of proteins that are already present in the cell. tie up the RNA and you won't get translation Repressor proteins and antisense RNA can inhibit translation. The translation of mRNA can be influenced by proteins that influence the ability of the ribosome to form a polypeptide. Translational regulatory proteins recognize sequences within the mRNA. These are called translational repressors. Translational repressors may inhibit translation by binding to the Shine-Dalgarno sequence, or by binding to a region of the mRNA that promotes an RNA secondary structure that inhibits translation. Antisense RNA is a piece of RNA that is complementary to the mRNA. An example is osmoregulation in E. coli (Figure 14.16), in which the production of antisense RNA inhibits translation of the mRNA. Posttranslational regulation can occur via feedback inhibition and covalent modifications. A common mechanism of regulation for metabolic enzymes is feedback inhibition (Figure 14.17). In this case the final product of the pathway inhibits the activity of one or more enzymes in the pathway. An allosteric enzyme has two different binding sites. The catalytic site is responsible for the binding of the substrate. The regulatory site allows for a means of turning the enzyme off, usually by a conformational change to the enzyme and catalytic site. ! $! Covalent modifi cation v ia Phosphorylation Enzymes may also be modified by covalent modification of their structure. This may include proteolytic processing, disulfide bond formation, or the attachment of sugars, functional groups or lipids to the enzyme. This process is known as posttranslational covalent modification. virus has invaded Gene Regulation in the Bacteriophage Life Cycle t he cell, going to use the materials We will take a very quick, abbreviated look at bacteriophage of the cell to make new virsues, and Phage ! can follow a lytic or lysogenic life cycle. t hen burst and release them life cycle gene regulation. During the lytic cycle, the genetic instructions of the bacteriophage direct the synthesis of many copies of the phage genetic material and coat proteins that are assembled to make new phages. When synthesis and assembly are complete, the bacterial host cell is lysed, and the newly made phages are released into the environment. In the lysogenic cycle, the phage integrates its genetic material into the bacterial chromosome, forming a prophage. In this state, it is replicated whenever the bacterial cell divides. At a later stage, a prophage may become activated to excise itself from the bacterial chromosome and enter the lytic cycle. The genome of phage ! is illustrated in Figure 14.18. Normally the genome is linear, until it is injected into the host bacterial cell. integrase on the left lysis proteins on t he right The genes on the left of the diagram are used in the lysogenic cycle. The genes on the right of the diagram are used in the lytic cycle. The genes at the top are used very soon after infection initiating a competition between the lysogenic and lytic cycles. The ! repressor protein controls the lysogenic cycle, the cro protein controls the lytic cycle, and the OR region provides a genetic switch between the lytic and lysogenic cycles (Figures 14.18 and 14.20). The OR region contains three operators. cro and ! repressor can bind to all three sites. These proteins determine the switch between the lytic and lysogenic cycles. there are actually 3 operators in the Or s ite lambda controls lysogenic and cro protein controls lytic ! %! Pr is for lytic cycle and PRM is for lysogenic cycle During the lysogenic cycle, the ! repressor controls the switch; during the lytic cycle the cro protein controls the switch. The ! repressor turns off PR and turns on PRM, sending the cycle to the lysogenic side. The cro protein turns off PRM and turns on PR, sending the cycle to the lytic side. Please see the Conceptual Summary and Experimental Summary for Chapter 14 on pages 383 and 384. This lecture outline was prepared from Genetics: Analysis and Principles, by Brooker, 2009 (3rd edition). It contains phrases and entire sentences taken verbatim from that source, and is in no way meant to represent original work by Mark Bierner. ! &! ...
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