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

typeSteady State Levels

Synthetic Capacity

Stability

rRNA 83% 58% High

tRNA 14% 10% High

mRNA 3% 32% 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 ’

• The ’ subunit is involved in DNA binding

• The subunit contains the polymerase active site

• The subunit acts as scaffold on which the other subunits assemble.

• Also requires -factor for initiation –forms holo enzyme complex

Site of DNA binding and

RNA polymerization

-factor• The -factor is required for binding of the RNA

polymerase to the promoter• Association of the RNA polynerase core complex w/

the -factor forms the holo-RNA polymerase complex

• W/o the -factor the core complex binds to DNA non-specifically.

• W/ the -factor, the holo-enzyme binds specifically with high affinity to the promoter region

• Also decreases the affinity of the RNA polymerase to non-promoter regions

• Different -factors for specific classes of genes

General Gene Structure• 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.

5’ 3’Transcribed region terminatorPromoter

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 -factor binds.

Other -Factors• Standard genes – 70

• Nitrogen regulated genes – 54

• Heat shock regulated genes – 32

How does RNA polymerase finds the

promoter?• RNA polymerase does not disassociate from DNA strand and reassemble at the promoter (2nd 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 PPi) • When 6-10 unit oligonucleotide has been

made, sigma subunit dissociates, completing "initiation“

• NusA protein binds to core complex after disassociation of -factor to convert RNA polymerase to elongation form.

Transcriptional Initiation

Closed complex

Open complex

Primer formation

Disassociation of -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

Eukaryotic RNA Polymerases

type Location ProductsRNA polymerase

I Nucleolus rRNA

RNA polymerase II

Nucleoplasm mRNA

RNA polymerase III

Nucleoplasm

rRNA, tRNA, others

Mitochondrial RNA polymerase

Mitochondria

Mitochondrial gene

transcripts

Chloroplast RNA polymerase

Chloroplast

Chloroplast gene

transcripts

Eukaryotic RNA Polymerases

• RNA polymerase I, II, and III

• All 3 are big, multimeric proteins (500-700 kD)

• All have 2 large subunits with sequences similar to and ' 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 and ' • RPB1 has DNA-binding site; RPB2 binds

NTP • RPB1 has C-terminal domain (CTD) or

PTSPSYS • 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

AAAAAA5’CAPmRNA

RNA Processing

RNA Degradation

Protein DegradationPost-translational modification

Activeenzyme

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 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 the ‘operator’

• 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)

-galactosidase – Cleaves lactose to glucose and galactose.

-galactoside transacetylase – acetylates -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.