Hon Molecular Exam 2

Chromatin, Bacterial Transcription, Eukaryotic Transcription

chromatin

DNA+Protein used as a packaging mechanism for chromosomes

bacterial chromatin

*nucleoid

• NAPs

• DNA Supercoiling

does bacterial chromatin have histones

no! they use NAPs as a substitute

eukaryotic chromatin

1. dna double helix

2. dna wrapped in histones → forms nucleosomes

3. nucleosomes coiled into a chromatin fiber

4. futher condensation

5. mitotic chromosome

histones

small basic proteins rich in Lysine (K) and Arginine (R)

• they are both basic and positively charged

• allows histones to interact with negatively charged phosphate backbone of DNA

Core histones

H2A, H2B, H3, H4

• 2 copies of each

A core nucleosome contains ~_____ bp of DNA wrapped around a histone octamer

146

H1

Linker histone, not part of core octamer

Lysine + Argenine make up more than ___ of histone composition (percentage)

20%

histone variants

• CENP-A → in centromeres → replaces H3 in nucleosomes, provides platform for kinetochore assembly and chromosome segregation

• H2AX → at sites of DNA Damage → phosphorylated at Ser139, marks DNA break regions and helps recruit repair factors

• H2AZ → inactive chromatin → associated with active gene expression

nucleosome

basic unit of eukaryotic chromatin (bundle of 8 histones)

centromere (specialized heterochromatin)

responsible for cells ability to properly segregate sister chromatids during cell division

• specifically linked to CENP-A

chromatin remodeling

uses energy from hydrolysis of ATP to alter chromatin structure

• uses large protein complexes

  • repositioning: slides nucleosome so DNA binding site is exposed

  • ejection: removes nucleosome

  • unwrapping: poses DNA (transient/reversible

  • dimer exchange: swaps a histone dimer (alters composition)

  • dimer ejection: removes a dimer (alters composition)

euchromatin (OPPOSITE FOR HETEROCHROMATIN)

transcription: active

nucleosome density: low, open, depleted

DNA Methylation: low

histone acetylation: high

histone methylation: low

H3K4 methylation: high (exception)

H3K9/H3K27 methylation: low

5-Methylcytosine

5-mc

generally associated with transcription repression

• methylated at transposable elements and gene body

• unmethylated at promoters

DNA methylation

adding a methyl group to suppress/turn off gene activity

• both in bacteria and eukaryotes

• protects against cleavage of their genomes by restriction enzymes

E.Coli methylation

E.Coli has a methyl transferase enzyme called DAM that methylates DNA

• methylates A in sequence GATC

types of histone covalent modifications

acetylation (KST)

methylation (KR)

phosphorylation (STYHD)

eukaryotic acetylation

KST

• frequently found at the promoters and enhancers of active genes → associated with activation of gene

  • lysine acetylated by histone acetyltransferases (HATs)

  • lysine removed by histone deacetylases (HDACs)

• neutralizes lysines positive charge → weaker histone = more accessible chromatin

eukaryotic methylation

KR

• H3K4 methylation → high in euchromatin, found at enhancers and promoters

• H3K9/H3K27 methylation → high in heterochromatin, found upstream of silent genes

• H3K36/H3K79 methylation → found at bodies of active genes

Creates binding sites for reglatory proteins, usually associated with repression

eukaryotic phosphorylation

STYHD

• H2AX: Phosphorylation on serine 139 is a characteristic of sites of DNA Breakage

• phosphorylation cascade

phosphorylation cascade

major means by which the cell responds to extracellular signals

• signal → kinase activation → phosphorylation of next protein → more phosphorylation → altered regulator activity → altered gene expression

x-inactivation

inactivation of one copy of the x chromosome in female cells

• one x chromosome is inactivated by XIST (functional noncoding RNA)

• XIST leads to the recruitment of factors that methylate H3K9/H3K27 and DNA

Murray Barr + Ewart Bertram 1948

in female cats, neurons found a darkly stained spot in the nucleus

• condensed inactive X is called a Barr body

Susumu Ohno 1959

one x-chromosome was darkly stained → marker of heterochromatin

• barr body should look like heterochromatin (transcriptionally repressed, high methylation, enriched for repressive histone methylation)

Mary Lyon 1961

proposed that inactivation is random

• mx and px are equally likely to be silenced → lyonization

hox gene

specify body plan in multicellular organisms

• present in every cell but expressed differently in each

  • must be expressed in the righ cell types and silenced everywhere else

polycomb gene (PcGs)

represses scr expression in middle + hind legs so sex combs only form on front legs of male flies

• found to catalyze H3K27me3 (methlation of H3K27 → repression)

trithorax gene

activates scr gene in front legs

• if mutated, scr expression is reduced in front legs

• catalyzes H3K4me3 which is an exception and is associated with active gene transcription

  • can also catalyze H3K27ac (acetylation of H3K27)

bacterial transcription basics

• first step in flow of genetic information

• DNA-Dependent RNAP

• catalyze formation of phosphodiester bonds → hydrolysis of phosphodiester bonds is unlikely because of pyrophosphate hydrolysis which releases a lot of free energy

  • (RNA-Dependent RNAP used for RNA interference not transcription)

• nascant RNA only grows 5’ to 3’

• RNAP's do not require a primer

Exception: reverse transcription by retroviruses (HIV)

RNAP core enzymes (sufficient for RNA synthesis)

a - complex assembly

a - complex assembly

b - forms “pincer”, contains rifampicin-binding pocket (inhibitor)

b’ - forms “pincer”, catalytic subunit

omega - complex assembly

RNAP holoenzymes

a - complex assembly

a - complex assembly

b - forms “pincer”, contains rifampicin-binding pocket (inhibitor)

b’ - forms “pincer”, catalytic subunit

omega - complex assembly

sigma - recognizing promoters (required for initiation), ejected during elongation

transcription promoters

• required, must be upstream

• consensus sequences: derived from alignments

  • -35

  • -10

• mutations: changing spacing between boxes usually decreases transcription but can potentially increase it

Methods for discovering promoter boxes

DNase Protection Assay

• incubate RNAP holoenzyme with a DNA fragment containing a promoter

• isolate protected fragment and determine its sequence

DNase 1 footprinting technique

• a bound protein leaves a footprint

  • gap in the cleavage ladder reveals the exact protected region

sigma factor

recognizes specific promoters and is required for the initiation of transcription

• sigma 70 aka sigma D : major sigma factor during exponential growth

• sigma 54 aka sigma N: nitrogen metabolism genes

bacterial initiation : RNAP promoter complex

1. RNAP holoenzyme recognizes promoter and forms a closed complex

   1. closed complex: DNA strand stays together

2. DNA melts are the staert site and forms the open complex

   1. open complex: DNA strand separates

      1. permanganate footprinting: attacks single-stranded DNA only

bacterial initiation continued

RNAP scrunches downstream DNA (8-10 nt)

• open complex formed

• accumulation of enough energy from scrunching allows promoter to escape → productive elongation

• sigma factor is ejected

bacterial elongation

NTPs reach catalytic site and provide the chemical energy for chain extension

• drives RNAP downstream

RNAP pauses during elongation because it does not always move at a fixed rate

• can pause and backtrack

• mismatched DNA is cleaved (cut): GreA and GreB enhace cleavage allowing a corrected 3’ end to re-enter active site and resume elongation

bacterial termination

Instrinsic

• GC-rich inverted repeat → RNA stem loop → causes RNA to pause

• approx 8-10 U-rich residues → improtant for release of nascent RNA chain

Rho-Dependent

• C-rich rho utilization (rut) site in RNA + Rho Factor

polycistronic mRNA

multiple protein-coding sequences with one promoter

• common in prokaryotes, rare in eukaryotes

monocistronic mRNA

single protein chain

• common in eukaryotes

operon

segment of DNA containing multiple genes regulated by a single promoter

• polycistronic

Jacques Monod + Francois Jacob 1960

performed experiments to understand how the genes in E.Coli allowing lactose to metabolize lactose are regulated

• used constitutive mutants to make lac mRNA regardless of presence of lactose

• they made partial diploids because E.Coli is haploid

  • found mutations LacI and LacO

lac operon composition

promoter, operator, LacZ, LacY, LacA

• lacZ, LacY, and LacA are in high levels with high lactose medium until they account for 1% of total protein in cell (Inducible)

• mutations: LacI, LacO

official order: laci, laco, lacz, lacy, laca

lacL (mutation)

encodes the diffusible LacI repressor

• recessive: partial diploids wiht one good copy of LacI showed inducible synthesis

• when lactose is present, LacI is not at operator

LacO^c (mutation)

operator

DNA site bound by LacI

• cis-acting regulatory element

• “c” = constitutive

• can cause constitutive synthesis of lac mRNA only when it is on the same DNA molecule as lacZ⁺

  • constitutive if it has LacO^c/LacZ⁺ somewhere ***

  • inducible if not

LacZ

B-Galactosidase

• breaks lactose into galactose and glucose

• inducible

LacY

Lactose permease, imports lactose

• inducible

LacA

B-galactoside transacetylase

CRP site

binds cAMP-CRP activator when glucose is low

• when glucose is present, cAMP-CRP activator is not

which is preferred: glucose or lactose

glucose!!

if there are high levels of Trp…

RNAP is terminated and mRNA is not extended

• does not want to make more tryptophan, wants to use from environment if possible

• ribosome does not stall at tandem trp codons → 3-4 terminator forms → terminates early → trp gene OFF

if there are low levels of trp…

• ribosome stalls at tadem Trp codon → 2-3 antiterminator forms → continues transcription → Trp gene ON

TrpR

on/off switch for repressor system

• decreases transcription ~70-fold

Attenuation

A sequence of 28 bases in the leader

• provides finer control

• decreases transcription ~10-fold

similarities between bacterial and eukaryotic transcription

• large multi-subunit RNAPs

• need a promoter for initiation

• regulated by DNA binding proteins

differences between bacterial and eukaryotic transcription

• eukaryotic transcription has multiple RNAPs

• eukaryotic has more initiation factors

• eukaryotic has nucleosomes (histones)

RNAP I (euk)

encodes rRNA

RNAP II (euk)**

encodes mRNA, incRNA, miRNA, etc…

RNAP III (euk)

encodes tRNA

RNAP VI (euk)

encodes siRNA (only in plants!)

eukaryotic protein-coding gene structure

regulatory promoter upstream of a core promoter, followed by 5’-exons and introns-3’

• exons: protein coding regions

• introns: non-protein coding regions that break up exons

core promoters

TATA box

BREs (2)

Inr

DPE

Assembly of PIC in correct order

core promoters: BRE - TATA box - BRE - Inr - DPE

GTFs: TFIID → A > b → F Pol II → E → H

TATA box

bound by TBP (subunit of TFIID) to initiate PIC formation

• only 10% of promoters in any eukaryotic genome have TATA boxes

BREs

TFIIB response elements, bind TFIIB

Inr

initiator element near the transcription start site

DPE

Downstream promoter element

TAFs

TBP-associated factors that allow TFIID to recognize Inr/DPE in absence of TATA box

TFIID

• contains TBP and 13 TAFs

• contains Inr and DPE

• scaffold for PIC, integrates regulatory signals

Regulation of TBP

T_1/2 > 1Hr

• persistent binding of TBP could result in over-activation

• solution: Mot 1 - enzyme that removes TBP from DNA

TFIIA

• after TFIID

• stabilizes binding of TBP and TFIID to the core promoter

TFIIB

• after TFIIA

• binds BREs

• stabilizing binding of TFIID to core promoter

• helps recruit the RNAP II - TFIIF complex (next)

• participates in selection of transcription start sites (TSSs)

TFIIF-Pol II

recruited along with RNAP II

• necessary for TFIIE/TFIIH recruitment

• participates in TSS selection

TFIIE

• after TFIIF

• helps recruit TFIIH

• necessary for promoter melting

TFIIH

• last GTF

• acts as helicase and unwinds DNA to form open complex

• phosphorylates RNAP II CTD

regulatory promoters

upstream of core promoters

bound by specific activators

regulatory enhancers

distance-independent

orientation-independent (forward or backward)

• contains specific protein-binding sequences

• acts by looping to target promoters

SV40

study of SV40 transcription revealed two identical sequences upstream of reg. promoter

• removal of one → small inhibition of transcription

• removal of both → transcription stopped completely

• enhancer is not specific not SV40

Can be inserted forwards OR backwards

insulators

• mediated by CTCF protein

• blocks enhacer activity and/or creates a boundary between euchromatin and heterochromatin

activators

two domains:

1. DNA-binding domain → recognizes specific sequences in promoters and enhancers

2. activation domain → contacts transcription machinery

uses two-hybrid assay in lab

mediator

bridge between transcription activators and RNAP

• required for euk transcription

• head and middle: contact RNAP II

• tail: contacts activator

reporter assays

used to test the strength and activity of a promoter

• common examples:

  • green flourescent protein (GFP)

  • LacZ (blue pigment used in cloning)

eukaryotic elongation

• C-terminal domain must be phosphorylated

• RNAP II has regulated pausing

  • regulated by NELF and DSIF (negative charge)

  • pause released by P-TEFb (positive charge)

• stalled/backtracked RNAP II is rescued by TFIIS

• nucleosomes represent impediments to RNAP II progress

  • FACT (facilitates chromatin transcription)

FACT

• allows RNAP II to transcribe through packaged DNA

• removes a single dimer of H2A-H2B ahead of RNAP→ loosens packaging

• reassembles nucleosome behind RNAP by returning missing histones

eukaryotic termination

RNAP I : TTF-1 recognizes a specific sequence in rRNA genes and blocks further transcription

RNAP II : No single specific termination seuqence mentioned ; requires RNA processing

RNAP III : Transcripts have ~4-7 Us at the 3- end → triggers RNAP III release