Chapter 7 Summary.docx

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Molecular Biology and Genetics
MBG 3080
Wendy Keenleyside

th I have the third edition textbook, so references to figures may vary if you have the 4 ed Chapter 7: Lytic Bacteriophages: Development, Genetics, and Generalized Transduction Bacteriophages (phages) – viruses that infect bacteria – most abundant biological entity – not a living organism but a nucleic acid (DNA or RNA) wrapped in a protein and/or membrane coat for protection The Bacteriophage Lytic Development Cycle To start infection, phage attaches to specific receptor on cell surface, it then injects its DNA into cell, where transcription of RNA (usually by host RNA polymerase) begins almost immediately. Not all genes transcribed to mRNA when DNA first enters cell, only some genes have promoters that mimic those of the host DNA and so are recognized by host RNA polymerase. These genes are called early genes, and they encode for enzymes involved in DNA synthesis (i.e. DNA polymerase, primase, DNA ligase, and helicase). With the help of these enzymes, the phage DNA begins to replicate and copies accumulate in cell. The rest of the genes (late genes) are transcribed. Promoters of late genes not recognized by host RNA polymerase. Most of these genes encode for the proteins involved in assembling the head and tail. After the phage is completed, DNA is taken up by the heads, and tails are attached – cells break open (lyse) –new phages released to infect other cells. Fig 7.2 Regulated by having genes be transcribed to mRNA only at certain times (transcriptional regulation) and sometimes posttranslational regulation. Posttranscriptional regulation is any regulation in the expression of a gene that occurs after the mRNA has been synthesized from the gene, for example in the rate of translation of mRNA. Translational regulation is the variation, under different conditions, in the amount of synthesis of a polypeptide due to variation in the rate at which the polypeptide is translated from the mRNA. One or more of the gene products synthesized during each stage of development turns on the transcription of the genes in the next stage – can also turn off transcription of genes expressed in the previous stage. This is called a regulatory cascade. Fig 7.3 Regulatory genes easily identified by mutations. Mutations in most genes affect only affect the product of the mutated gene; in regulatory genes the mutation affects the expression of many other genes. Phage T7: a Phage-Encoded RNA polymerase Only early and late genes. Expression goes from left to right: Antirestriction-Protein kinase-RNA polymerase-DNA ligase-(late genes) DNA metabolism-phage assembly. Transcription of late genes helps pull DNA into cell, causing sequential gene expression. T-7 specific RNA polymerase. Fig 7.4 pET vectors – (plasmid expression T7) Gene for T7 RNA polymerase (gene 1) is inserted into E. coli chromosome and transcribed from lac promoter – expressed only if the inducer IPTG is added. Gene 1 transcribes the gene cloned into pET vector downstream of the T7 late promoter on the pET cloning vector. The T7 lysozyme encoded by a compatible plasmid, pLysS, binds to any residual T7 RNA polymerase made in the absence of induction and inactivates it. The presence of lac operators between the T7 promoter and the cloned gene further reduced transcription of the cloned gene in the absence of IPTG. Fig 7.5 Phage T4: Transcriptional Activators, Antitermination, a New Sigma Factor, and Replication- Coupled Transcription Delayed-early genes transcribed from sigma 70 promoter (same as immediate early genes) but regulated by anti-termination mechanism. Without some T4 regulatory gene products, the RNA polymerase which has initiated at an immediate-early promoter will stop before it gets to the delayed-early. This means that transcription of these genes must await the synthesis of antitermination factors encoded by the phage. Middle gene products transcribed from middle-mode promoters – diff from sigma 70 promoters in that their -35 sequence is replaced by a sequence centered at -30 called a Mot. They different so transcription from these promoters requires the phage-encoded MotA and AsiA proteins. AsiA protein binds to region 4 of sigma 70 and inhibits its binding to the -35 sequence. Once bound to sigma 70, AsiA allows MotA to bind, this allows MotA to recognize the -30 sequence of the middle T4 promoters. True-late genes-last to be transcribed. Products are mostly the head, tail and tail fiber components of the phage and enzymes and proteins needed to lyse the cell. Initiation of T$ late genes coupled to replication of DNA. T4 transcribed by promoters with sequence TATAAATA (not -10 sequence), and they lack a -35 sequence. This means host RNA pol. Doesn’t normally recognize the T4 promoters. Product of T4 regulatory gene 55 solves this, it’s an alternative sigma factor that binds to host RNA pol and changes its specificity, so that it recognizes on promoters from T4 late genes. Gene 55 only recognizes altered -10 sequence, and therefore cant form a open complex and initiate transcription. Needs gp 33 (gp55/gp33 split sigma factor) to bind to where region 4 of sigma 70 would bind (on RNA pol). gp33 initiates transcription only when it has bound to the sliding clamp gp45, which allows open complex formation and the initiation of transcription. gp45 sliding clamp: clamp loaders gp44 and gp62 load gp45 clamp on dsDNA (can also load at nick in DNA). Clamp stays on DNA when DNA pol comes off and then it can slide along the DNA in either direction. If clamp comes in contact with gp33 bound to RNA pol at a late promoter, it allows the RNA pol to initiate transcription. Phage DNA Replication DNA pol can’t initiate synthesis of new
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