Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33MCB 320 - spring semesterMechanisms of Human DiseaseAdvanced MCB credit - 3 semester hoursOverview:Instructors:Eric Bolton, Ph.D. [email protected] Llano, M.D., Ph.D. [email protected]: Undergraduate Juniors and Seniors with biomedical interestsPrerequisite:MCB 252 or instructor consent Coleman and Tsongalis, 2009, Molecular Pathology: The Molecular Basis of Human DiseaseLearn how molecular and cellular defects manifest as pathologies that affect the function of human tissues and organs. This course focuses on the pathophysiology of common human diseases and the environmental, genetic, and epigenetic causes of specific disease types. Disease topics include cardiovascular diseases, stroke, cancer, asthma, COPD, diabetes, metabolic syndrome, neurologic disorders, and reproductive disorders.Propagation of bacteriophage X ,Part IViruses are molecular parasites of cellular life•Like cells, a virus has a nucleic acid genome that encodes its proteins. Viral genomes can be RNA or DNA, and single- or double-stranded. •Unlike cells, a virus cannot reproduce in isolation. To propagate it must infect a living cell and commandeer the host’s internal biochemistry.•Cells of different species have distinct viral parasites. Viruses that infect bacteria are known as 'bacteriophages'.For a bacteriophage, the initial steps of infection are binding of the viral particle to the host cell surface, and injection of the viral genome across the membrane into the cytoplasm.NOTEBacteria have evolved defense mechanisms - e.g. restriction enzymes; the CRISPR locus [lecture 36] - that can potentially destroy bacteriophages which enter the cell.For today’s lecture we will ignore these defenses, and focus instead on what happens to the phage when infection and propagation are successful.Bacteriophagelambda (w)headtailDNAChromosome:•48.5 kb ds-DNA•71 protein-coding genes •Linear chromosome has complementary 5' overhangs at each end. Inside the bacterium, these comple-mentary sequences anneal and the chromosome is circular.Since its discovery in 1950, w phage has been one of the most widely used systems in the study of molecular biology, and also as a vector for cloning DNA.Bacteriophage life cyclesThis is a 2-way genetic switch - i.e. during infection, each phage must commit to either lysogeny or lysis.Lysogenic CycleLysogenic CycleLytic CycleLytic CycleWatson, Fig. 18-20Following bacterial infection, the phage chooses between lysis and lysogeny by adopting one of two distinct patterns of gene regulation.Watson, Fig. 18-21Genome map: bacteriophageUltimately the decision between lysis and lysogeny is determined by the transcriptional activity at 4 promoters in this 5 kb control region.Circularization siteThere are two genes in the control region, cI and cro, whose protein products compete with one another to decide whether the phage becomes lytic or lysogenic.•cro is the key gene for lysis.•cI is the key gene for lysogeny. Note that cro and cI are transcribed in opposite directions, i.e. they use different template strands of the DNA.Lysis vs. lysogeny: critical genes<<<<<<<>>>>>>>chromosome Lysis vs. lysogeny: promotersWatson: Fig. 18-30•PL and PR are strong constitutive promoters, i.e. RNA polymerase alone is sufficient for active transcription. •They control 2 polycistronic operons that extend in opposite directions around the chromosome. TRANSCRIPTIONTRANSCRIPTIONWatson, Fig. 18-30 PRM and PRE are weak promoters, i.e. RNAP requires activator proteins to efficiently transcribe from either of these promoters.When they are active, either promoter can be used to transcribe the cI gene. Lysis vs. lysogeny: promoterscIII cI cro cII>>>>>> >>>>>><<<<<<<<<<<<TRANSCRIPTIONchromosomeWhat happens when w infects a cell?Watson: Fig. 18-30The strong PL and PR promoters recruit the bacterial RNA polymerase and initiate a high level of transcription. This includes expression of the cro gene. If this continues unchecked, the phage will enter the lytic cycle.TRANSCRIPTION'TXN'cIII cI cro cII>>>>>> >>>>>><<<<<<<<<<<<chromosomeWatson, Fig. 18-31However, transcription from the PR promoter also leads to synthesis of the cII protein, a transcription factor which serves as an activator at the weak PRE promoter.Active transcription from PRE leads to expression of the cI gene.>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<There are two genes in the control region, cI and cro, whose protein products compete with one another to decide whether the phage becomes lytic or lysogenic.•cro is the key gene for lysis.•cI is the key gene for lysogeny. Note that cro and cI are transcribed in opposite directions, i.e. they use different template strands of the DNA.Lysis vs. lysogeny: critical genes<<<<<<<>>>>>>>XREPRESSEScro cIREPRESSESBoth cro and cI encode transcription factors, and both of them can repress the other’s transcription.Both Cro and cI proteins can bind to three operator sequences (OR1-OR3) that overlap with the PRM and PR promoters:NOTE: although not shown here, there is a symmetric set of operators (OL3-OL1) situated adjacent to the PL promoter.Cro protein has its highest affinity for the OR3 operator.When bound to OR3, Cro blocks the PRM promoter and represses cI transcription from this promoter. The PR promoter isn’t blocked, and remains active.<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>ONWatson, Fig. 18-27On the contrary, cI protein (also known as the w repressor) has its highest affinity for the OR1 operator. When bound to OR1, cI blocks the PR promoter and represses cro transcription.<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>OFF= cI proteinPRMPRWatson, Fig. 18-27ONXREPRESSEScro cIREPRESSESBoth cro and cI encode transcription factors, and both of them can repress the others transcription.Who wins?Affinity forCI proteinLow (1X) Low (1X)