Molecular Biology Topics for Second Part of Molecular Biology Course: Structural Organization of Eukaryotic Chromosomes Spring 2009 BI 425 UOG

NonTranscribed genes are less susceptible to Dnase 1 Digestion

Fluorescent Labeled Probes hybridized to Interphase Chromosomes

Prokaryotic Gene Regulation

Deletion Analysis: Identify Upstream Transcription Control Sequences

Determination of Consensus TATA Box Sequence

Scanning Mutations Identify Transcription Control Elements

General Pattern of Control Elements that Regulate Gene Expression in Yeast and other Eukaryotes.

LIFE CYCLE OF HIV-1 RNA DNA DNA Provirus Latency Viral Transcription Viral RNA RNA Processing mRNA Viral Proteins Viral genomic RNA Virion Assembly Maturation & Release of Virus HIV Virus 3’ LTR pol env nef if vpr vpu rev Shubir Ghosh, UCSF

Overview of Eukaryotic Transcriptional Control

Column Chromatography Separation of the three RNA Polymerases In Eukaryotes.

Transcription Assay: Transcription Start Site

EMSA

Dnase 1 Footprinting

Dnase1 Footprint Assay in Transcription Factor Purification

Northern Blot & S1 Nuclease Assay: Identify Enhancers of Transcription S1 Nuclease Assay Northern Blot Homework!

In vitro Transcription Assay: Identification of Transcription Factors

In vivo Transfection Assay: Identify & Evaluate Transcription Factors

Eukaryotic Transcriptional Activators: Features & Experimental Analysis

Eukaryotic Transcriptional Activators: Features & Experimental Analysis of GAL4

Eukaryotic Transcriptional Activators: Features & Experimental Analysis of GAL4

Interaction of bacteriophage 434 Repressor with DNA

Interaction of Zinc Finger proteins with DNA GR

Interaction of Lecuine Zipper & basic HLH proteins with DNA Yeast Gcn4 Transcription Activator

Cooperative binding of two unrelated transcription factors to neighboring sites in a composite control element

Combinatorial Possibilities due to formation of Heterodimeric Transcription Factors

Transcriptional Repression : Yeast mating Locus

Antibody and DNA probes colocalize RepressorProtein, Sir3, with telomeric heterochromatin in Yeast nuclei Telomeres labeled by Hybridization to Telomere Specific DNA probe (Yellow) Telomere specific DNA Hybridization Probe Fluorescent labeled Antibody specific For Sir3 protein

Schematic Model of Silencing Mechanism at Yeast Telomeres Dependence of Silencing on Hypoacetylation of the Histone Tails

Handout on Histone Acetylation/Deacetylation! Model of Transcriptional Activation Mechanism See HACs & HDACs

Chromatin Immunoprecipitation Assay: Evaluate State of Histone Acetylation on Nucleosome

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:

Mechanism of Transcriptional Activation by Gcn4

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

Mechanism of Transcriptional Repression

Medistor Complex in Euksryotic Transcription