Chapter 3.

DNA, RNA, and Protein

Synthesis

4. Transcription

Gene Expression

RNA

Polymerase

Gene (ORF)

Transcription

Factor

DNA

mRNA

Transcription

Translation

Protein

Regulatory region (promoter)

5’ flanking region

Upstream region Coding region

5’ UTR 3’ UTR

3’ flanking region

Downstream region

Terminator

RNA

Structure Ribose: both 2’ and 3’ OH

Uracil(U) instead of thymine (T)

Mostly single strand

Secondary structure Intrastrand base-pairing

Composition of RNA in metabolically active cellmRNA: 3~5 %

rRNA: 90%

tRNA: 4%

Types of RNA

RNA Polymerase

RNA synthesis using ssDNA as a template

Prokaryote One RNA polymerase

Eukaryote RNA Pol I rRNA (28S, 5.8S, 18S)

RNA Pol II mRNA

RNA Pol III tRNA, 5S rRNA

5’ to 3’ RNA synthesis

RNA Polymerase

OFR1 OFR2 OFR3-35 -10 Operator

mRNA

promoter

Terminator

Genes

Gene

Specific nucleotide sequence that is transcribed into RNA

Structural gene Gene encoding protein

Polycystronic transcription Prokaryote only

Transcription of multiple genes in one transcription unit

mRNA Structure

Genes Can Be Transcribed with Different

Efficiencies

Three Phases of Transcription

Initiation

:Binding of RNA polymerase to template DNA

Elongation

: Synthesis of RNA (5’ to 3’, NTP)

Termination

: Release of the enzyme and RNA from the DNA

template

a2bb’w

Catalytic subunits

for RNA

polymerization

s

Determine

promoter

specificity

Holoenzyme (Core enzyme)

Prokaryotic RNA polymerase

TTGACA TATAAT Gene (ORF)

-35 Box -10 Box

+1

20 to 600 nt

s

Transcription Initiation in Prokaryotes

a2bbw

TTGACA TATAAT

AACTGT ATATTA

5’

3’ 5’

3’

TTGACA

AACTGT

5’

3’ 5’

3’TAT

ATA

-35 -10

Closed

Complex

Open

Complex 5’ 3’

Coding, Sense

Anticoding, Antisense

Template

s

s

a2bb

a2bb

pppA or pppG

Transcription Initiation in Prokaryotes

Transcription Cycle of Prokaryotic RNA

polymerase

Directions of Transcription

Transcription Elongation and Termination

DNA unwinding: ~17 bp

~12 bp

Transcription Termination in Prokaryotes (1)

ρ(rho) -independent terminator

Region of dyad symmetry centered about 20 - 30

bases upstream of the last nucleotide in the

transcript.

Absolute sequence is not conserved, but the hairpin

structure is conserved.

A string of ~6 U's in RNA product

UUUUUU5’

1. Formation of the hairpin causes

RNA polymerase to pause.

2. Formation of the hairpin in the

nascent RNA limits the

RNA/DNA hybrid to a short

segment of dA/U base pairs

3. Nascent RNA is released,

DNA/DNA hybrid re-forms, and

RNA polymerase falls off

Model for the mechanism of

rho-independent termination

Transcription Termination in Prokaryotes (2)

ρ(rho) -dependent terminator

Hairpin structure similar to rho-independent

terminators

Lack the poly-U stretch

Require the activity of a rho-protein, a trans-acting

factor that functions as a homohexamer

1. Rho translocates 5' to 3' along nascent

RNA

2. Rho binds consensus sequence (rut :

rho utilization) located approximately

100 bases upstream of the

transcriptional termination site

3. RNA polymerase pauses at hairpin

4. Rho unwinds DNA/RNA hybrid

(RNA-dependent ATPase activity)

Model for the mechanism of

rho-dependent termination

Regulation of Transcription Initiation in

Prokaryotes

Different s factors

Recognize different consensus sequences

e.g. E. coli s70 s54 s32 ss

Transcription factors

Activators

Repressors

Negative supercoiling

Gyrase (Topoisomerase II)

generates negative supercoiling

Topoisomerase I

Relaxes excess negative supercoiling

Regulation of mRNA Transcription in

Prokaryotes

Negative controlled system

Repressor Repression in the

presence of effectormolecule

Repression with a coreporessor

Trp Repressor

Regulation of mRNA Transcription in

Prokaryotes

Positive controlled system

Activator Promoting RNA polymerase activity

Activated or repressed by effectors

Repressors and Activators

5. Transcription in Eukaryotes

Components of Transcriptional Control in

Eukaryotes

Cis elements: Regulatory DNA sequences

Promoter

RNA polymerase binding site (TATA box)

Promoter-proximal elements

Enhancer (eukaryotes only)

Trans elements: Specific proteins binding to the

regulatory sequence

RNA polymerase complex

RNA polymerase

General transcription factors

Mediators

Transcription factors

Activators

Repressors

Promoter Elements in Eukaryotic Transcription

TATA box: direct RNA polymerase

25-35 bp upstream of TC start site

Promoter proximal elements: 10 to 20 bp

< 200 bp of TC start site

Enhancer: Composed of multiple elements of 10-20 bp, 100 bp long

Upto 50 kb upstream or downstream, or within an intron

RNA Polymerase: E.coli vs. Yeast

Polymerase Genes transcribed

RNA Pol I Ribosomal RNA genes (28S, 5.8S, 18S)

RNA Pol II Protein-coding genes

Most small nuclear RNA (sn RNA) genes

RNA Pol III Genes for tRNA

5S ribosomal RNA

U6-sn RNA

small nucleolar (sno) RNA

18S rRNA

30 different proteins

60S

40S

Ribosome

28S, 5.8S, and 5S rRNA.

45 different proteins

RNA Polymerase in Eukaryotes

Pre-initiation complex (PIC) of RNA Pol II

RNA Polymerase II

2 Large subunits

10 small subunits

General transcription factors

(TFIIA), TFIIB, TFIID, TFIIE, TFIIF, and TFIIH

TBP (TATA-box binding protein)

TAFs (TBP associated factors)

Assembly of PIC

TBP

Regulation of Transcription initiation

Mediator

Mediate activated transcription by binding to

transcription factors and RNA Pol II

SWI/SNF

Chromatin remodeling

SAGA

Histone acetyltransferase (HAT) complex

Chromatin Remodeling and Histone

Modification

Transcription Initiaion

Ordered Assembly of Transcription

Initiation Complex

Molecular Mechanisms of Transcription

Activation and Repression

Modulation of Chromatin structure

Chromatin remodeling

Acetylation and deacetylation:

Acetylation of Histone H3, H4

Unstructured chromatin

Facilitate assembly of transcription initiation complex

Heterochromatin:

Condensed chromain, telomeres, centromeres

Euchromatin:

most transcribed genes are located

Interaction with Pol II and general transcription factors

Overview of Transcription Control in

Multicellular Eukaryotes

Mechanism of Histone Deacetylation in

Yeast Transcriptional Control

Mechanism of Histone Acetylation in

Yeast Transcriptional Control

Promotor Exon ExonIntron

-25 +1

Inr

Primary mRNA

Transcription

Splicing

AAAAA7mG

Translation

mRNA

AAAAA

Eukaryotic Structural Gene

t

7mG

Exon: 150~ 200 bp

Intron: 40 ~ 10,000 bp

Processing of mRNA

5’ Capping

Splicing

3’ Polyadenylation

•5’ end blocked by the

addition of a 7-

methylguanosine.

•The cap structure has no

free phosphates

•Protected from attack by

phosphatases, or

nucleases

5’ Capping

Polyadenylation

•The polyadenylation sequence is

bound by a cleavage and

polyadenylation specificity factor

(CPSF).

•An endonuclease cleaves the

transcript.

•PolyA polymerase binds to CPSF,

adding up to 250 adenylate

residues to the 3' end of the

transcript.

Consensus Sequence for Splicing in Humans

Pre mRNA Splicing

Mechanisms Splicing by Spliceosome

Alternative Splicing

Different pattern of exon splicing from a primary transcript

Generation of tissue-specific proteins from the same structural gene

The Export of mRNA Through the

Nuclear Pore