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

ViralTranscription

Viral RNA

RNA Processing

mRNA

Viral Proteins

Viral genomic RNA

Virion Assembly

Maturation &Release of Virus

HIV Virus

Shubir Ghosh, UCSF

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

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 byHybridization to TelomereSpecific DNA probe (Yellow)

Telomere specificDNA Hybridization Probe

Fluorescent labeledAntibody specificFor 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