Transcription: Eukaryotes and Prokaryotes

Transcription: DNA to RNA

I. Eukaryotes

A. Types of RNA polymerase

1. RNA polymerase I genes transcribed

-5.8S, 18S, 28S rRNA genes

2. RNA polymerase II genes transcribed

-all protein-coding genes

-snoRNA genes

-some snRNA genes

3. RNA polymerase III genes transcribed

-5S rRNA genes

-snRNA genes

-genes for other small RNAs

B. Initiation and Transcription Factors

1. The TBP (TATA binding protein) of TFIID binds to the TATA box

30 nucleotides 5’ of the start of transcription in the promoter. TFIID

binds the start of transcription and the DPE site 30 nucleotides

downstream of the start site.

2. TFIIA and TFIIB bind the promoter. TFIIB binds the BRE site 35

nucleotides 5’ of the start of transcription.

3. TFIIF binds RNA polymersase II

4. TFIIE and TFIIH bind the promoter. TFIIH is a DNA helicase and Protein kinase that phosphorylates the c-terminal tail of RNA polymerase II allowing the polymerase to disengage transcription factors.

5. An Activator protein binds the Enhancer region 5’ of the promoter

6. A Mediator protein binds to the RNA polymerase II and transcription factors and to the Activator protein

7. Histone acetylase and the chromatin remodeling complex bind the Mediator protein to facilitate transcription of DNA associated with nucelosomes.

C. Elongation

1. RNA polymerase II associates with elongation factors shortly after the

start of transcription. They rescue stalled polymerases and help it

navigate through chromatin. (the exact proteins are unknown).

2. Superhelical tension is created as the polymerase moves down DNA

(positive tension in front of it and negative tension behind it). DNA

topoisomerases covalently bind to the DNA backbone. It creates a

single stranded nick by creating a break in the phosphodiester

backbone allowing the DNA to swivel.

D. RNA Modification

Capping factors, splicing factors and polyadenylation factors bind the c-terminal RNA polymerase II tail.

1. 5’ Capping

-After RNA Polyermase II produces about 25 nucleotides of nacent mRNA three enzymes acting sequentially add a 5’ cap consisting of a modified guanine nucleotide. The enzymes are:

a. Phosphatase

-Removes the 5’ phosphate

b. Guanylyl transferase

c. Methyl transferase

2. RNA splicing

-The Spliceosome removes intervening introns.

Components of the spliceosome are small nuclear RNAs

(snRNA). ATP hydrolysis is required for the assembly

and rearrangements of the spliceosome but not the actual

chemical reaction of RNA splicing. The sequence of

events are:

a. BBP and U2AF bind the 3’ branch point site

b. U2 snRNP displaces BBP and U2AF and forms

base pairs with the consensus sequence.

c. U1 snRNP forms base-pairs with the 5’ splice

site

d. U4/U6 and U5 enter the spliceosome. U4 and U6

are held together by base pairing.

e. RNA-RNA rearrangements break apart the

U4/U6 base pairing and a lariat is formed. U4 is

ejected from the spliceosome and U6 displaces

U1 at the 5’ splice site that is cleaved to expose a

free OH.

f. RNA rearrangements occur that activating the

spiceosome. The 3’splice site is cleaved joining

the two exon sequences together.

-Alternate forms of Splicing include

a. AT-AC Sliceosome

-1% of splicing

-Recognizes a different set of 5’ and 3’ sequences

-RNA-RNA interactions are the same

-U11 recognizes the 5’ site and U12 recognizes

the 3’ site

b. Trans (common in Trypanosomes)

-A single exon is spliced on the 5’ end of many

different RNA transcripts.

-SL RNP, U4/U6, and U5 recognize the 3’ end of

the 5’ exon and U2 recognizes the 5’ end of the

3’ exon.

3. 3’ Polyadenylation

a. CPSF (cleavage and polyadenylation specificity factor)

and CstF (cleavage stimulation factor F) bind to the c-

terminal tail of RNA polymerase II.

b. As the nacent mRNA strand moves through the RNA

polymerase exit tunnel, CPSF and CstF bind specific

sequences on the RNA strand (AAUAAA).

c. Additional proteins assemble with CPSF and CstF to

cleave RNA creating a 3’end.

d. PAP (poly-A-polyermase) adds approximately 200

Adenosines at the cleavage site one at a time using ATP

as a precursor. PAP does not require a template.

e. Poly-A-binding proteins bind to the tail and by an

unknown mechanism determine the length of the tail.

They remain attached to the tail as mRNA is transported

from the nucleus to the cytoplasm and helps direct

protein synthesis on ribosomes.

E. Repression of Transcription: Gene Repressor Proteins

-There are Five different ways that eukaryotic gene repressor proteins can operate.

1. Competitive DNA binding

-Gene Activator and Gene Repressor proteins compete for binding to the

same regulatory DNA sequence

2. Masking the activation surface

-Gene activator and Gene repressor proteins bind separate DNA binding s

site but the Gene Repressor protein binds the active site of the Gene Activator

3. Direct interaction with the general transcription factors

-The Gene Repressor protein can interact with transcription factors early

and prevent further assembly or can interact late and inhibit disassembly

of transcription factors from the RNA polymerase during elongation

4. Recruitment of repressive chromatin remodeling complexes

-The Gene Repressor protein recruits repressive chromatin remodeling

complexes converting the promoter region to its pre-transcriptional state.

5. Recruitment of histone deacetylases

-.Histone deacetylase reduces the affinity of TFIID for the promoter and

decreases the accessibility of DNA

II. Prokaryotes

A. RNA Polymerase (RNAP)

1. RNAP core enzyme

a. Alpha Subunit (2)

-Chain initiation

-Interaction with regulatory proteins and upstream

promoter elements

b. Beta Subunit

-Chain initiation

-Chain elongation

c. Beta’ Subunit

-DNA binding

d. Theta Subunit

-Unknown

2. RNAP holoenzyme

a. RNAP core enzyme

b. Sigma Factor

3. Ecoli Sigma factors

a. Sigma 28 (sigma F)

-gene: fliA

-regulation of flagellar expression

b. Sigma 24 (sigma E)

-gene: rpoE

-Regulation of heat shock and oxidative stress

c. Sigma 38 (sigma S)

-gene: rpoS

-Growth-phase regulated proteins

d. Sigma 70 (sigma D)

-gene: rpoD

-Proteins produced at high temperature

e. Sigma 54 (sigma N)

-gene: rpoD

-Regulation of nitrogen and fermentation and metabolism

f. Sigma 32 (Sigma H)

-gene: rpo H

-Proteins produced at high temp

g. Sigma 19 (sigma I)

-gene: fecI

-Proteins produced in response to ferric acid

B. Initiation

1. Binding of the sigma subunit converts RNAP from a closed form to an

open form allowing the enzyme to bind a DNA template

2. RNAP cruises down the DNA strand until it reaches the promoter site

which is recognized by the sigma factor.

3. RNAP binds tightly to DNA (mediated by the sigma factor). DNA is

wrapped around the RNAP (approximately 75-80bp and 7-8 helical turns).

4. RNAP converts to a closed conformation: Closed Promoter Complex.

5. DNA in the active site of the RNAP melts for 10-15bp: Open Promoter

Complex.

6. The first few nucleotides of RNA are synthesized.

C. Elongation

1. The sigma subunit is released once RNA is about 10nts long

2. The RNAP core enzyme loses contact with the promoter region and continues synthesizing RNA

3. Once the polymer is 15-20 nucleotides in length, the RNAP is only contacting 30-40 nucleotides.

4. New nucleotides are added to the strand at 45 nucleotides per second

5. At about 5 nucleotides from the RNAP active site the RNA emerges from the enzyme and can fold into secondary structures.

6. Other proteins involved in elongation include

a. NusA (elongation factor)

-Binds to the RNAP core and may replace the sigma factor

-increases K’s for NTPs (retards chain elongation at low

NTP levels and accentuates pusing at natural pause sites)

b. NusG (elongation factor)

-Enhances elongation rate

-May act to convert RNAP paused conformation into

elongation formation

c. GreA (antiarrest factor)

-Induces cleavage near the 3’ end of stalled or arrested

transcription complexes

-Cleaved 3’ end fragments dissociate and RNAP resumes

elongation from the new 3’ terminum

-release di- or trinucleotides

-must interact with transcription complex before arrest

d. GreE (antiarrest factor)

-Acts similarly to GreA, but cleaves oligonucleotides up to

9 nucleotides in length

-Can rescue stalled complexes.

D. Termination: Two mechanisms

1. Rho-indepent termination

a. Transcription of terminator sequence

-GC rich region 20 nucleotides preceding 8 A’s in the template

strand

b. Sequence forms a stem-loop structure once it emerges from the RNAP

and signals the RNAP to stop transcription

2. Rho-dependent termination

a. Requires

-Rho protein

-TSP (transcriptional stop point)

-RUT (Rho-utilization site)

b. Rho binds the rut site in mRNA

c. Rho moves to the end of the mRNA strand

-Requires ATP hydrolysis

d. Leads to the dissociation of RNA:DNA duplexes when 3’ end of RNA

is paired.

e. NusG protein is required for efficient termination

-may allow Rho to interact with the transcription complex shortly

after initiation so that Rho can scan along RNA for the rut site

E Regulation of Transcription

1. Initiation

a. Use of different sigma factors

b. Anti-sigma factors

-bind to sigma factors and block their ability to interact with

RNAP core enzyme, blocking initiation of transcription

-ex. Sigma 28 encodes along with several flagellar genes, an anti-

sigma 28 protein

c. Repression of initiation: lac operon

-LacI tetramer is bound to promoter region blocking RNAP access

-Allolactose (inducer) binds to LacI tetramer, LacI releases from

the operator and give RNAP access.

d. Activation of Initiation: lac operon

-Low glucose stimulates adenylate cyclase, increasing cAMP

which binds to CRP

-CRP-cAMP binds to the CAP site in the lac promoter

-CRP-cAMP bends DNA 90 degrees stimulating a closed complex

formation

-CRP-cAMP interacts with RNAP alpha subunit

2. Elongation

A. Example: Tryptophan operon

1. There is a leader peptide coding region that encodes multiple

tryptophan residues.

2. Three potential stem loops can form downstream of the leader

peptide coding region depending on how fast the RNAP is

transcribing DNA

-1to2 loop

-3to4 loop

-2to3 loop

3. Following possible stem loop structures is a polyU tract (rho-

independent terminatior)

B. During LOW Tryptophan levels

1. Ribosome stalls at Tryptophan codons in leader peptide

(remember transcription and translation are tightly coupled in

prokaryotes)

2. 2to3 stem loop forms blocking 3to4 terminator loop

3. Transcription proceeds

C. During HIGH Tryptophan levels

1. Ribosome easily translates the leader peptide, occluding region

to

2. Formation of 3to4 terminator loop

3. Poly U tract is transcribed (rho-independent terminator)

4. RNAP falls off

3. Termination

A. Example: Bacteriophage lambda growth

1. The bacteriophage grows either as a lytic phage enters lysogeny

a. During lytic growth Early to Middle gene expression

specifies DNA replication and structural gene expression

-Requires lambda N gene

b. Middle to Late gene expression specifies bacteriophage

assembly and host cell lysis

-Requires lambda Q gene

c. Anti-Terminator proteins are:

1. N protein

-Acts at NutL and NutR sites

2. Q protein

-Acts as Qut site

3. Requires host proteins

a. NusA

b. NusB

c. NusG

d. Ribosome protein S10

e. Rho