Chapter 21 (part 1)

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.