Transcription

Central Dogma

Genes

• Sequence of DNA that is transcribed. • Encode proteins, tRNAs, rRNAs, etc.. • “Housekeeping” genes encode proteins or

RNAs that are essential for normal cellular activity.

• Simplest bacterial genomes contain 500

to 600 genes.

• Mulitcellular Eukaryotes contain between

15,000 and 50,000 genes.

Types of RNAs

• tRNA, rRNA, and mRNA • rRNA and tRNA very abundant

relative to mRNA.

• But mRNA is transcribed at higher

rates than rRNA and tRNA

• Abundance is a reflection of the

relative stability of the different forms of RNA

RNA Content of E. coli Cells

type Steady State Levels Synthetic Capacity Stability rRNA 83% 58% High tRNA mRNA 14% 3% 10% 32% High Very Low

Phases of Transcription

• Initiation: Binding of RNA polymerase to

promoter, unwinding of DNA, formation of primer.

• Elongation: RNA polymerase catalyzes

the processive elongation of RNA chain, while unwinding and rewinding DNA strand

• Termination: termination of transcription

and disassemble of transcription complex.

E. Coli RNA Polymerase

• RNA polymerase core

enzyme is a multimeric protein

a 2 ,b, b

’

, w • The • The b

’ subunit is involved in DNA binding site

• The b a

subunit contains the polymerase active subunit acts as scaffold on which the other subunits assemble.

• Also requires s

-factor for initiation –forms holo enzyme complex Site of DNA binding and RNA polymerization

s

-factor

• The s

-factor is required for binding of the RNA polymerase to the promoter

• Association of the RNA polynerase core complex

w/ the

s

-factor forms the holo-RNA polymerase complex

• W/o the s

-factor the core complex binds to DNA non-specifically.

• W/ the

region

s

-factor, the holo-enzyme binds specifically with high affinity to the promoter

• Also decreases the affinity of the RNA

polymerase to non-promoter regions

• Different s

-factors for specific classes of genes

5’

General Gene Structure

Promoter Transcribed region • Promoter – sequences

recognized by RNA polymerase as start site for transcription.

• Transcribed region –

template from which mRNA is synthesized

• Terminator –

sequences signaling the release of the RNA polymerase from the gene.

terminator 3’

Gene Promoters

• Site where RNA polymerase binds and initiates

transcription.

• Gene that are regulated similarly contain

common DNA sequences (concensus sequences) within their promoters

Important Concensus Sequences

• Pribnow Box – position –10 from

transcriptional start

• -35 region – position –35 from

transcriptional start.

• Site where s 70

-factor binds.

Other

s

-Factors

• Standard genes – s

70

• Nitrogen regulated genes – s • Heat shock regulated genes –

54

s

32

How does RNA polymerase finds the promoter?

• RNA polymerase does not disassociate

from DNA strand and reassemble at the promoter (2 nd order reaction – to slow)

• RNA polymerase holo-enzyme binds to

DNA and scans for promoter sequences (scanning occurs in only one dimension, 100 times faster than diffusion limit)

• During scanning enzyme is bound non-

specifically to DNA.

• Can quickly scan 2000 base pairs

Transcriptional Initiation

• Rate limiting step of trxn. • Requires unwinding of DNA and synthesis

of primer.

• Conformational change occurs after DNA

binding of RNA polymerase holo-enzyme.

• First RNA Polymerase binds to DNA

(closed-complex), then conformational change in the polymerase (open complex) causes formation of transcription bubble (strand separation).

Initiation of Polymerization

• RNA polymerase has two binding sites for NTPs • Initiation site prefers to binds ATP and GTP (most

RNAs begin with a purine at 5'-end)

• Elongation site binds the second incoming NTP • 3'-OH of first attacks alpha-P of second to form a

new phosphoester bond (eliminating PP i )

• When 6-10 unit oligonucleotide has been made, sigma

subunit dissociates, completing "initiation“

• NusA protein binds to core complex after

disassociation of

s

-factor to convert RNA polymerase to elongation form.

Transcriptional Initiation

Closed complex Open complex Primer formation Disassociation of

s

-factor

Chain Elongation

Core polymerase - no sigma

• Polymerase is accurate - only about 1

error in 10,000 bases

• Even this error rate is OK, since many

transcripts are made from each gene

• Elongation rate is 20-50 bases per

second - slower in G/C-rich regions (why??) and faster elsewhere

• Topoisomerases precede and follow

polymerase to relieve supercoiling

Transcriptional Termination

• Process by which RNA polymerase

complex disassembles from 3’ end of gene.

• Two Mechanisms – Pausing and

“rho-mediated” termination

Pausing induces termination

• RNA polymerase can stall at

“pause sites”

• Pause sites are GC rich

(difficult to unwind)

• Can decrease trxn rates by

a factor of 10 to 100.

• Hairpin formation in RNA

can exaggerate pausing

• Hairpin structures in

transcribed RNA can destabilize DNA:RNA hybrid in active site

• Nus A protein increases

pausing when hairpins form.

3’end tends to be AU rich easily to disrupt during pausing. Leads to disassembly of RNA polymerase complex

Rho Dependent Termination

• rho is an ATP-

dependent helicase

• it moves along RNA

transcript, finds the "bubble", unwinds it and releases RNA chain

Eukaryotic Transcription

• Similar to what occurs in

prokaryotes, but requires more accessory proteins in RNA polymerase complex.

• Multiple RNA polymerases

type

Eukaryotic RNA Polymerases

Location Products RNA polymerase I RNA polymerase II RNA polymerase III Mitochondrial RNA polymerase Chloroplast RNA polymerase Nucleolus Nucleoplasm Nucleoplasm Chloroplast rRNA mRNA rRNA, tRNA, others Chloroplast gene transcripts

II, and III

Eukaryotic RNA

• All 3 are big,

(500-700 kD) subunits with

Polymerases

• RNA polymerase I,

multimeric proteins

• All have 2 large

sequences similar to

b

and

b

' in E.coli RNA polymerase, so catalytic site may be conserved

Eukaryotic Gene Promoters

• Contain AT rich concensus sequence

located –19 to –27 bp from transcription start (TATA box)

• Site where RNA polymerase II binds

RNA Polymerase II

• Most interesting because it regulates

synthesis of mRNA

• Yeast Pol II consists of 10 different

peptides (RPB1 - RPB10)

• RPB1 and RPB2 are homologous to E. coli

RNA polymerase PTSPSYS

b

and

b

'

• RPB1 has DNA-binding site; RPB2 binds NTP • RPB1 has C-terminal domain (CTD) or • 5 of these 7 have -OH, so this is a

hydrophilic and phosphorylatable site

More RNA Polymerase II

• CTD is essential and this domain may

project away from the globular portion of the enzyme (up to 50 nm!)

• Only RNA Pol II whose CTD is NOT

phosphorylated can initiate transcription

• TATA box (TATAAA) is a consensus

promoter

• 7 general transcription factors are

required

Transcription Factors

• Polymerase I, II, and III do not bind

specifically to promoters

• They must interact with their promoters

via so-called transcription factors

• Transcription factors recognize and

initiate transcription at specific promoter sequences

Transcription Factors

• TFAIIA, TFAIIB –

components of RNA polymerase II holo enzyme complex

• TFIID – Initiation factor,

contains TATA binding protein (TBP) subunit. TATA box recognition.

• TFIIF – (RAP30/74)

decrease affinity to non promoter DNA

Eukaryotic Transcription

• Once initiation complex assembles

process similar to bacteria (closed complex to open complex transition, primer formation)

• Once elongation phase begins most

transcription factor disassociate from DNA and RNA polymerase II (but TFIIF may remain bound).

• TFIIS – Elongation factor binds at

elongation phase. May also play analogous role to NusA protein in termination.

Transcriptional Regulation and RNA Processing

Gene Expression

• Constitutive – Genes expressed in

all cells (Housekeeping genes)

• Induced – Genes whose expression

is regulated by environmental, developmental, or metabolic signals.

Regulation of Gene Expression

Active enzyme

Post-translational modification RNA Processing

5’CAP

mRNA

AAAAAA

RNA Degradation Protein Degradation

Transcriptional Regulation

• Regulation occurring at the initiation of

transcription.

• Involves regulatory sequences present

within the promoter region of a gene (cis-elements)

• Involves soluble protein factors (trans-

acting factors) that promote (activators) or inhibit (repressors) binding of the RNA polymerase to the promoter

Cis-elements

• Typically found in 5’ untranscribed

region of the gene (promoter region).

• Can be specific sites for binding of

activators or repressors.

• Position and orientation of cis

element relative to transcriptional start site is usually fixed.

Enhancers

• Enhancers are a class of cis-elements

that can be located either upstream or downstream of the promoter region (often a long distance away).

• Enhancers can also be present within the

transcribed region of the gene.

• Enhancers can be inverted and still

function 5’-ATGCATGC-3’ = 5’-CGTACGTA-3’

Two Classes of Trans Acting Factors

• Activators and

repressors- Bind to cis-elements.

• Co-activators and

co-repressors – bind to proteins associated with cis elements. Promote or inhibit assembly of transcriptional initiation complex

Structural Motifs in DNA-Binding Regulatory Proteins

• Crucial feature must be atomic contacts between

protein residues and bases and sugar-phosphate backbone of DNA

• Most contacts are in the major groove of DNA • 80% of regulatory proteins can be assigned to one

of three classes: helix-turn-helix (HTH), zinc finger (Zn-finger) and leucine zipper (bZIP)

• In addition to DNA-binding domains, these proteins

usually possess other domains that interact with other proteins

The Helix-Turn-Helix Motif

• contain two alpha

helices separated by a loop with a beta turn

• The C-terminal helix

fits in major groove of DNA; N-terminal helix stabilizes by hydrophobic interactions with C terminal helix

The Zn-Finger Motif

Zn fingers form a folded beta strand and an alpha helix that fits into the DNA major groove.

The Leucine Zipper Motif

• Forms amphipathic

alpha helix and a coiled-coil dimer

• Leucine zipper proteins

dimerize, either as homo- or hetero dimers

• The basic region is the

DNA-recognition site

• Basic region is often

modeled as a pair of helices that can wrap around the major groove

Binding of some trans-factors is regulated by allosteric modification

Transcription Regulation in

the ‘operator’

Prokaryotes

• Genes for enzymes for pathways are grouped in

clusters on the chromosome - called operons

• This allows coordinated expression • A regulatory sequence adjacent to such a unit

determines whether it is transcribed - this is

• Regulatory proteins work with operators to

control transcription of the genes

Induction and Repression

• Increased synthesis of genes in response

to a metabolite is ‘induction’

• Decreased synthesis in response to a

metabolite is ‘repression’

lac operon

• Lac operon – encodes 3 proteins involved

in galactosides uptake and catabolism.

• Permease – imports galactosides (lactose)  b  b

-galactosidase – Cleaves lactose to glucose and galactose.

-galactoside transacetylase – acetylates

b

-galactosides

• Expression of lac operon is negatively

regulated by the lacI protein

The lac I protein

• The structural genes of the lac operon are

controlled by negative regulation

• lacI gene product is the lac repressor • When the lacI protein binds to the lac operator

it prevents transcription

• lac repressor – 2 domains - DNA binding on N-

term; C-term. binds inducer, forms tetramer.

Inhibition of repression of lac operon by inducer binding to lacI

• Binding of inducer to lacI cause allosteric change that

prevents binding to the operator

• Inducer is allolactose which is formed when excess

lactose is present.

Catabolite Repression of lac Operon (Positive regulation)

• When excess glucose is present, the lac operon

is repressed even in the presence of lactose.

• In the absence of glucose, the lac operon is

induced.

• Absence of glucose results in the increase

synthesis of cAMP

• cAMP binds to cAMP regulatory protein (CRP)

(AKA CAP).

• When activated by cAMP, CRP binds to lac

promoter and stimulates transcription.

Post-transcriptional Modification of RNA

• tRNA Processing • rRNA Processing • Eukaryotic mRNA Processing

tRNA Processing

•tRNA is first transcribed by RNA •Polymerase III, is then processed •tRNAs are further processed in the chemical

modification of bases

rRNA Processing

•Multiple rRNAs are originally transcribed as single

transcript.

•In eukaryotes involves RNA polymerase I •5 endonuclases involved in the processing

Processing of Eukaryotic mRNA

5’ Capping

• Primary transcripts (aka pre-mRNAs or

heterogeneous nuclear RNA) are usually first "capped" by a guanylyl group

• The reaction is catalyzed by guanylyl

transferase

• Capping G residue is methylated at 7-

position

• Additional methylations occur at 2'-O

positions of next two residues and at 6 amino of the first adenine

• Modification required to increase mRNA

stability

3'-Polyadenylylation

• Termination of transcription occurs only

after RNA polymerase has transcribed past a consensus AAUAAA sequence the poly(A)+ addition site

• 10-30 nucleotides past this site, a

string of 100 to 200 adenine residues are added to the mRNA transcript the poly(A)+ tail

• poly(A) polymerase adds these A

residues

• poly(A) tail may govern stability of the

mRNA

Splicing of Pre-mRNA

• Pre-mRNA must be capped and polyadenylated before

splicing

• In "splicing", the introns are excised and the exons are

sewn together to form mature mRNA

• Splicing occurs only in the nucleus • The 5'-end of an intron in higher eukaryotes is always

GU and the 3'-end is always AG

• All introns have a "branch site" 18 to 40 nucleotides

upstream from 3'-splice site

Splicing of Pre-mRNA

• Lariat structure forms

by interaction with 5’splice site G and 2’OH of A in the branch site.

• Exons are then joined

and lariot is excised.

• Splicing complex

includes snRNAs that are involved in identification of splice junctions.