molecular biology topics for second part of molecular biology course: structural organization of...
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Molecular Biology
Topics for Second Part of Molecular Biology Course:
Structural Organization of Eukaryotic Chromosomes
Spring 2009 BI 425 UOG
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NonTranscribed genes are less susceptible to Dnase 1 Digestion
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Fluorescent Labeled Probes hybridized to Interphase Chromosomes
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Prokaryotic Gene Regulation
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Deletion Analysis: Identify Upstream Transcription Control Sequences
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Determination of Consensus TATA Box Sequence
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Scanning Mutations Identify Transcription Control Elements
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General Pattern of Control Elements that Regulate Gene Expression in Yeast and other Eukaryotes.
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LIFE CYCLE OF HIV-1
RNA
DNA
DNA Provirus
Latency
ViralTranscription
Viral RNA
RNA Processing
mRNA
Viral Proteins
Viral genomic RNA
Virion Assembly
Maturation &Release of Virus
HIV Virus
Shubir Ghosh, UCSF
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TRANSCRIPTIONAL REGULATION OF HIV-1
5’ LTR 3’ LTR
TAT
+ 1- 119 - 80 - 30
TBP Sp1 NF-kBTAR
+ 80
+ 1 - 80 - 30
TBP Sp1TAR
+ 80
+ 1- 119 - 80 - 30
TBP TATA
Sp1 NF-kB TAR
+ 80
USFNF-AT
- 453
AP-1
TATA
TATA
Shubir Ghosh, UCSF
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Overview of Eukaryotic Transcriptional Control
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Column Chromatography Separation of the three RNA Polymerases In Eukaryotes.
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Transcription Assay: Transcription Start Site
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EMSA
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Dnase 1 Footprinting
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Dnase1 Footprint Assay in Transcription Factor Purification
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Northern Blot & S1 Nuclease Assay:Identify Enhancers of Transcription
S1 Nuclease Assay Northern Blot
Homework!
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In vitro Transcription Assay: Identification of Transcription Factors
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In vivo Transfection Assay: Identify & Evaluate Transcription Factors
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Eukaryotic Transcriptional Activators: Features & Experimental Analysis
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Eukaryotic Transcriptional Activators: Features & Experimental Analysis of GAL4
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Eukaryotic Transcriptional Activators: Features & Experimental Analysis of GAL4
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Interaction of bacteriophage 434 Repressor with DNA
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Interaction of Zinc Finger proteins with DNA
GR
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Interaction of Lecuine Zipper & basic HLH proteins with DNA
Yeast Gcn4 Transcription Activator
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Cooperative binding of two unrelated transcription factors to neighboring sites in a composite control element
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Combinatorial Possibilities due to formation of Heterodimeric Transcription Factors
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Transcriptional Repression : Yeast mating Locus
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Antibody and DNA probes colocalize RepressorProtein, Sir3, with telomeric heterochromatin in Yeast nuclei
Telomeres labeled byHybridization to TelomereSpecific DNA probe (Yellow)
Telomere specificDNA Hybridization Probe
Fluorescent labeledAntibody specificFor Sir3 protein
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Schematic Model of Silencing Mechanism at Yeast Telomeres
Dependence of Silencing on Hypoacetylation of the Histone Tails
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Handout on Histone Acetylation/Deacetylation!
Model of Transcriptional Activation Mechanism
See
HACs & HDACs
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Chromatin Immunoprecipitation Assay: Evaluate State of Histone Acetylation on Nucleosome
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Transcriptional Activation by Gcn4 in Saccharomyces cerevisae
Gcn4
UAS
Gcn5 + Multisubunit Complex
Gcn4 Activated Gene in S.c
Components of Gcn4 activated gene in yeast:
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Mechanism of Transcriptional Activation by Gcn4
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Mechanism of Transcriptional Repression by Ume6 in Yeast
Components of Repression in yeast by Ume6:
Ume6 Repressed gene in S.c
Ume6
Multisubunit complex including:
Sin3
Rpd3
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Mechanism of Transcriptional Repression
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Medistor Complex in Euksryotic Transcription
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