Gene regulation overviewand

Saccharomyces cerevisiae cell-type specific mating type promoters

Nate Sotuyo

Allison Suarez

Transcription

Prokaryotes

Overview

• (RNA)n + ribonucleoside triphosphate ⇌ (RNA)n+1 +PPi– Divalent metal ion required (Mg2+ orMn2+)

• Cytoplasm, mRNA isnot modified• 5 Steps

– Pre-initiation– Initiation– Promoter clearance– Elongation– termination

DNA Synthesis comparisonSimilarities• 5’ 3’• Nucleophilic attack by 3’ OH on phosphate• Driven by hydrolysis or pyrophosphate

– Pyrophosphate orthophosphate

Differences• No Primer required• Error rate higher

Pre-initiation

• RNA holoenzyme α2ββ'σω binds DNA– “slides” along double

helix

• Unwinds the DNA to form initiation bubble

Initiation and promoter clearance

Initiation• RNA polymerase binds promoter (with sigma

factor)– Prinow box (-10), -35 region

• First phosphodiester bond formedPromoter clearance• Abortive initiation• CTD phosphorylation by TFIIH

Elongation and Termination

Elongation• 5’3’, TU• σ factor released • TopoisomeraseII feedbackTermination• Rho-independent

– G-C hairpin followed by string of U’s

• Rho-dependent– ρ destabilizes mRNA-template interaction

Review: Gene Regulation and Transcription in Eukaryotes

• Similar to gene regulation in prokaryotes, except more complex: more types of regulatory proteins and interactions with regulatory regions in DNA

• Both prokaryotes and eukaryotes have promoters– Region of DNA that is the RNA polymerase’s transcription initiation site

• Important differences in eukaryotes:• Nucleosomes, chromatin

– Chromatin remodeling

• 3 types of RNA polymerases• RNA modification

– Transcription occurs in the nucleus– Translation occurs in the cytoplasm

• Binding of DNA-binding proteins outside the promoter region

Example of chromatin remodeling

• Chromatin remodeling is the changing of nucleosome position• Histones

– Core octamer, amino-terminal ends

– Covalent modification termed “histone code”

• Acetylation, deacetylation, and gene expression– Acetylation of certain histone residues can cause the histone to

slide along the DNA, no longer inhibiting a promoter region or other important DNA-protein binding sites necessary for transcription

http://www1.ci.uc.pt/yss/Material/pdf_RNA%20Regulation/figurea6.jpg

Overview of transcription translation in eukaryotes

Genomic Footprinting of the Promoter Regions of STE2 and

STE3 genes in the Yeast Saccharomyces cerevisiae

Brigitte Ganter, Song Tan and Timothy J. Richmond

Overview

Overall goal– Develop high resolution map of the

promoter regions of STE2 and STE3 – Analyze the chromatin structure in the

promoter regions using DMS methylation, micrococcal nuclease and DNase I.

Mating types review

• Three cell types– Haploid: a,α– Diploid: a/α

• Specific gene expression determined by MATa or MATα

• Three transcription regulators expressed– MATa1,MATα1,MATα2

• Other key transcription factors– MCM1, STE12, SSN6, TUP1

Transcription factors

• MCM1– Activates both a and alpha– Purified vs. crude extracts (Q factors?)– a-specific genes conformational change

• STE12– Component of pheromone response pathway– Binds PRE signaling cascade

• Poor interaction unless MCM1 present

• MATα2 – Precise positioning of nucleosomes downstream STE2 UAS

elements in α cells

Transcription factors cont.

• SSN6 and TUP1– Required for full repression of a-genes in

alpha cells– Mutated ssn6 resulted in alpha cells that

could mate with other alpha cells (Though not efficiently)

Haploid Mating overview

STE2

STE3

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=p-box&rid=mcb.section.3752#3756

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=p-box&rid=mcb.section.3752#3756

Diploid Mating overview

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=p-box&rid=mcb.section.3752#3756

Significant nucleosome positioning

• Contribute to transcription regulation

• Stably positioned nucleosomes STE2/ α cells– Only MCM1 bound no

stable array– TFIID binding site is

protected

• a-cells only protected in TATA region– Due to TFII binding, not

nucleosome

Nucleosomal protection cont.

• STE 3 more complicated– a-cells

• MCM1 binding to P’-box on coding strand only in vitro– in vivo result consistent with each other

– alpha-cells• Concerted binding of MCM1 and MAT-alpha1

– Additional protection not detected in vitro observed on Q-box side

• Possible other factors that bind to these regions

Genomic Footprinting Techniques

“Conventional PCR is not immediately applicable to sequencing or footprinting because it requires two defined ends. A sequence or footprint ladder is composed of a population of related nucleic acid fragments. One end of each fragment is fixed by a primer or restriction cut and is therefore the same for all, whereas the other end is determined by variable chemical cleavage or chain termination and is therefore unique for each fragment. To apply PCR to a sequence ladder, [Mueller and Wold] introduced a simple ligation step that adds a common oligonucleotide sequence to the unique end of each member. A primer complementary to this new common sequence is then used, together with a primer complementary to the original fixed end, for simultaneous exponential amplification of all members of the sequence ladder. The procedure has high selectivity and specificity that are derived from the design of the ligation step and choice of primers. It also has high fidelity; a footprint consists of subtle differences in the starting concentrations of particular members of a sequence a sequence ladder, and these differences are reproducibly retained through amplification.”

Mueller, P.R. and Wold, B. (1989). In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science, 246, 280-286

Ligation Mediated PCR

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Mueller, P.R. and Wold, B. (1989). In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science, 246, 280-286