BIO353 Lecture9 (Transcription SHF 2021 Long

BIO353 – lecture 9: Transcription – the primary process
the central dogma in Molecular Biology
BIO353.01 – Molecular Genetics shf – 2022

RNA transcription: cellular diversity
liver cell brain cell
same genome
(same genes)
hepatocyte-specific neuron-specific
gene expression gene expression
gene a gene x
gene b gene y
gene c gene z

RNA transcription: differences in gene expression (cell types)
TReg cells naïve T cells
expression
high
genes
low

RNA transcription: differences in gene expression (evolution)
Brain Heart Kidney Liver Testes
higher expression
in humans
relative expression
genes
higher expression
in chimpanzee

RNA transcription: differences / similarities with replication
base
sugar

sugar

base

RNA transcription: general overview
transcription
bubble
DNA non-template strand
direction of transcription
template strand
RNA

RNA transcription: general overview
RNA polymerase
transcription
bubble
DNA non-template strand
template strand
RNA

RNA transcription: where to start and where to end transcription ?
Bacteria
gene A gene B gene E gene G
5’ 3’
3’ 5’
gene C gene D gene F gene H
direction of transcription 1.000 bp
Eukaryotes
template strand
gene A for gene B gene C
5’ 3’
3’ 5’
template strand gene B
for gene A 10.000 to 100.000 bp

RNA transcription: simultaneous transcription
The same gene can be transcribed simultaneously multiple times.
gene 1 gene 2
RNA DNA
Transcription end Transcription end

start start

RNA transcription: where to start and where to end transcription ?
gene
transcription start site

(TSS)
RNA pol
coding strand
5’ 3’
3’ 5’
template strand
promoter terminator

RNA polymerase: structure
prokaryotic
b’
a w
Thermus aquaticus Lobster claw

RNA polymerase: structure (evolutionary conservation)
prokaryotic eukaryotic
b’
a w
Thermus aquaticus Saccharomyces cerevisiae

RNA polymerase: structure (evolutionary conservation)
prokaryotic eukaryotic
b
active
center
a Mg2+ ion
b’
a w
Thermus aquaticus Saccharomyces cerevisiae

subunits
two
copies
two RNA polymerase: structure (evolutionary conservation and divergence)
everything rRNAs mRNAs tRNAs

lncRNAs snRNAs
5S rRNA

RNA transcription: three phases of the transcription cycle
promoter recognition
initiation assembly of the RNA pol complex
melting of template DNA
promoter escape
elongation synthesis of the full-length transcript
RNA modifications (capping / splicing)
recognition of the transcription stop

termination polyadenylation

RNA transcription: three phases of the transcription cycle
promoter recognition
initiation assembly of the RNA pol complex
melting of template DNA
abortive transcription of 6 - 10 nt
promoter escape
elongation synthesis of the full-length transcript
RNA modifications (capping / splicing)
recognition of the transcription stop

termination polyadenylation

RNA transcription: initiation - promoter structure (E. coli)
araBAD GGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTT
araC GCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTG
bioA TTCCAAAACGTGTTTTTTGTTGTTAATTCGGTGTAGACTTGTAAACCTAAATCTTTT
bioB CATAATCGACTTGTAAACCAAATTGAAAAGATTTAGGTTTACAAGTCTACACCGAAT
galP2 ATTTATTCCATGTCACACTTTTCGCATCTTTGTTATGCTATGGTTATTTCATACCAT
lac ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
lacl CCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTC
rrnA1 AAAATAAATGCTTGACTCTGTAGCGGGAAGGCGTATTATCACACCCCCGCGCCGCTG
rrnD1 CAAAAAAATACTTGTGCAAAAAATTGGGATCCCTATAATGCGCCTCCGTTGAGACGA
rrnE1 CAATTTTTCTATTGCGGCCTGCGGAGAACTCCCTATAATGCGCCTCCATCGACACGG
tRNATyr CAACGTAACACTTTACAGCGGCGCGTCATTTGATATGATGCGCCCCGCTTCCCGATA
trp AAATGAGCTGTTGACAATTAATCATCGAACTAGTTAACTAGTACGCAAGTTCACGTA
11-15 nt 5-8 nt
TCTTGACAT TATAAT A 51
42 38 82 84 79 64 53 45 41 79 95 44 59 51 96 T
7
-35 element -10 element
C
(Pribnow box) G 42
TSS
(transcription start site)

RNA transcription: initiation - prokaryotic promoter structure

RNA transcription: initiation - prokaryotic promoter structure

RNA transcription: initiation - s factor and promoter recognition
RNA pol core enzyme
RNA pol holoenzyme
s factor

RNA pol holoenzyme
enzyme core
(a2, b, b’, w)
s70 factor


transcription start



RNA transcription: initiation and isomerization (promoter melting)
closed complex open complex
isomerization
binding of RNA pol RNA pol melts

to promoter DNA at the promoter

RNA transcription: s factor and isomerization (promoter melting)
closed complex intermediate step

(promoter not melted) (interactions weakened)
s2 s2
flipepd-out
bases from -10 element
à weakened interaction

RNA transcription: s factor and isomerization (promoter melting)

(promoter not melted) (promoter melted)
s2 s2

RNA transcription: s factor and base flipping
s bound to promoter open complex

RNA polymerase: molecular structure and function

open complex
• What is the size of the transcription bubble ?
• How can it be determined experimentally ?
RNA pol melts

DNA at the promoter

DNA topology: coils and twists
Lk = Tw + Wr
Tw: twist (# of times one strand wraps around the other)

Wr: writhe (# of times the double strand wraps around itself)
Lk: linking number (# of times one strand would need to pass through the other to unwind the DNA)


unbound RNA pol
change in superhelicity
nick,
relax,
ligate
# of active polymerase
units per genome
binding of RNA pol melts DNA adopts

RNA pol to promoter DNA at the promoter superhelical form

what is the exact position of the replication bubble ?

hydrogen bonds
melt
no base pairing
dimethyl
sulfate
methylated A base
dimethyl
bind RNA pol sulfate
promoter sequence
remove
RNA pol
limited
gel digestion
electrophoresis with S1
nicks
ssDNA

RNA pol melts the promoter between +2 and -11
fragments from S1-digested promoter DNA
R: RNA pol
S: S1 nuclease (nicks ssDNA)
Siebenlist U, RNA polymerase unwinds an 11-base pair segment of a phage T7 promoter, Nature, 1979, 279(5714):651-2
araBAD GGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTT
araC GCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTG
bioA TTCCAAAACGTGTTTTTTGTTGTTAATTCGGTGTAGACTYGYAAACCTAAATCTTTT
bioB CATAATCGACTTGTAAACCAAATTGAAAAGATTTAGGTTTACAAGTCTACACCGAAT
galP2 ATTTATTCCATGTCACACTTTTCGCATCTTTGTTATGCTATGGTTATTTCATACCAT
lac ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
lacl CCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTC
rrnA1 AAAATAAATGCTTGACTCTGTAGCGGGAAGGCGTATTATCACACCCCCGCGCCGCTG
rrnD1 CAAAAAAATACTTGTGCAAAAAATTGGGATCCCTATAATGCGCCTCCGTTGAGACGA
rrnE1 CAATTTTTCTATTGCGGCCTGCGGAGAACTCCCTATAATGCGCCTCCATCGACACGG
tRNATyr CAACGTAACACTTTACAGCGGCGCGTCATTTGATATGATGCGCCCCGCTTCCCGATA
trp AAATGAGCTGTTGACAATTAATCATCGAACTAGTTAACTAGTACGCAAGTTCACGTA
transcription bubble
11-15 nt 11 7 5-8 nt
TCTTGACAT TATAAT A 51
42 38 82 84 79 64 53 45 41
s3/4 linker
79 95 44 59 51 96 T
7
-35 element -10 element
C
(Pribnow box) G 42
TSS
(transcription start site)

RNA transcription: initiation and elongation
RNA pol
motor
RNA pol
complex
template strand
promoter
+1

RNA transcription: initiation and elongation (promoter escape)
CTP, GTP, UTP
-pol
ATP + pol
RNA pol makes

abortive transcripts
of 6 – 10 nt length
Carpousis AJ, Gralla JD, Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry, 1980, 19:3245-53.

RNA transcription: initiation and elongation (promoter escape)
transient excursion
inchworming
scrunching

RNA transcription: stepwise elongation and proofreading
RNA pol

RNA pol pauses / slows down
P-P-P-

RNA pol
non-template DNA
misincorporated
nucleotide template DNA
+ PP NTP
nascent RNA
1. - pyrophosphorylitic editing (kinetic proofreading)

RNA pol
non-template DNA
misincorporated
nucleotide template DNA
cleavage
nascent RNA
1. - pyrophosphorylitic editing (kinetic proofreading)
2. - backtracking and hydrolytic cleavage (requires GreB / elongation stimulation factor)


RNA transcription: stepwise elongation and DNA topology
moving RNA pol complex

RNA transcription: termination in bacteria - Rho-independent
mutations that disrupt

hairpin formation / termination


RNA pol
push / tension
pull / release

RNA transcription: termination in bacteria - Rho factor
Rho factor
non-template DNA
template DNA
RNA pol
nascent RNA

Rho
non-template DNA
rut site template DNA
RNA pol
transcribed rut site

nascent RNA

non-template DNA
Rho RNA pol

nascent RNA

non-template DNA
Rho RNA pol

progression / ATP hydrolysis
nascent RNA

RNA transcription: multiple RNA polymerases in eukaryotes

RNA transcription: eukaryotic promoter structure
prokaryotic promoter
eukaryotic promoter
B recognition element TATA box initiator downstream downstream

(GC box) core elements promoter element

RNA transcription: eukaryotic promoter structure
binding sites for general transcription factors
B recognition element TATA box initiator downstream downstream

(GC box) core elements promoter element

RNA transcription: assembly of general transcription factors
binding of TFIID / TBP at TATA box
recruitment of TFIIA and TFIIB

binding of TFIID / TBP at TATA box
recruitment of TFIIA and TFIIB
loading of RNA pol II

recruitment of TFIIE and TFIIH
TFIIH
formation of pre-initiation complex
melting of DNA à transcription bubble
TFIIH pumps DNA into RNA pol II à melting

recruitment of TFIIE and TFIIH
formation of pre-initiation complex

melting of DNA à transcription bubble
phosphorylation of CTD à initiation
à promoter escape

TFIID (includes TBP and TAFs)
order of recruitment
TFIIA promoter recognition
TFIIB asymmetry
TFIIF complex stabilization
Pol II
TFIIE complex stabilization
TFIIH promoter melting
DABFPEH = pre-initiation complex (PIC)


TATA-containing promoters
TBP
TFIID = TBP + TAFs
TBP + TAF1 / TAF2

TATA-less promoter TATA-less promoter

TATA-containing promoters with Inr and DPE with GC box

RNA transcription: additional factors required for transcription
upstream activator sequences / enhancer elements
proximal core promoter

RNA transcription: mediator complexes

RNA transcription: mediator complexes
upstream activator sequences / enhancer elements
proximal core promoter

RNA transcription: RNA pol I
rRNAs
rRNAs

RNA transcription: RNA pol III
tRNAs / 5S rRNA

RNA transcription: RNA pol III
size copy number

ORF1 ORF2 (pol)
LINEs autonomous AAA 6 – 8 kb 850.000 21%
A B
SINEs non-autonomous AAA 100 – 300 bp 1.500.000 13%
gag pol env

autonomous 6 – 11 kb
retrovirus-like 1.500.000 8%
gag
non-autonomous 1.5 – 3 kb
transposase
autonomous 2 – 3 kb
DNA transposons 300.000 3%
non-autonomous 80 – 2.000 bp

RNA transcription: association of C-terminal domain and RNA transcript
transcription
bubble
non-template strand
template strand
RNA pol II
nascent RNA

RNA transcription: association of C-terminal domain and RNA transcript
transcription
bubble
non-template strand
C-terminal domain template strand
RNA pol II
nascent RNA

RNA transcription: C-terminal domain structure
yeast plants mammals

RNA transcription: C-terminal domain - modifications
Y: tyrosine
S: serine
T: threonine
P: proline

TFIIH
à dissociation from mediator

TEFb
à elongation / recruitment of splicing factors



H3K4me
H3K36me

RNA transcription: polymerase pausing
RNA pol
density

RNA transcription: histones impede transcription
Laybourn PJ, Kadonaga JT, Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. Science, 1991, 254: 243.

RNA transcription: histones impede transcription

RNA transcription: post-transcriptional processing
TRANSCRIPTION nucleus
• 5’-capping
post-transcriptional
• splicing
modifications
• poly-adenylation
• RNA editing
• nuclear export
TRANSLATION cytoplasm

RNA processing: 5’ capping

RNA processing: 5’ capping

7metG-cap: function of the cap structure
Function of the cap:
• increased stability
• increased translation
• increased nuclear export
• increased splicing

the cap structure protects the RNA transcript from 5’ to 3’-degradation
mRNA in test tube mRNA extracted from oocytes
• capped
○ blocked
△ naked
long medium short

RNA length
long medium short

RNA length
Xenopus ocyte assay
Furuichi Y, et al., 5'-Terminal structure and mRNA stability. Nature. 1977, 266(5599):235-9.
the cap structure protects the RNA transcript from 5’ to 3’-degradation
time
0 0.5 1.0 1.5 2.0 (h) t1/2 (min)
Luc-A(-) 31
Luc-A(50) 44
cap-Luc-A(-) 53
cap-Luc-A(50) 100
Firefly (Photinus pyralis)
Gallie DR, The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev, 1991, 5(11):2108-16.

the cap structure increases translation of mRNAs
uncapped capped

the cap structure increases translation of mRNAs

polyA tail: not encoded in genomic DNA
>hba1_gene
...TTGAATAAAGTCGGAGTGGGCAGCAGCCTGTGTGTGTGCC
>hba_transcript
...TTGAATAAAGTCGGAGTGGGCAGCAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
polyA tail (~200 A)

>hba2
TTTGAATAAAGTCTGAGTGGGCAGCAGCCTGTGTGTGTGCC
>hba1
TTGAATAAAGTCGGAGTGGGCAGCAGCCTGTGTGTGTGCC
>nfkb1
ATAATAAAGGAAAAGCAAATTGTATGACCTCACCTTGTTTGTCTGTGCA
polyA signal 15 – 30 nt cleavage site GT-rich sequence
spacer

polyA tail: function of polyadenylation
:Function of the poly-A tail
• protection from degradation
• increased translatability
• facilitated nuclear export
• increased splicing

polyA-tail protects mRNA from degradation
Xenopus oocyte essay

polyA-tail increases mRNA translatability
polyA
Hela cells
Munroe D, Jacobson A, mRNA poly(A) tail, a 3' enhancer of translational initiation. Mol Cell Biol. 1990, 10(7): 3441–3455.

polyA-tail increases mRNA translatability by forming polysomes
mRNA ribosomes polypeptides
Munroe D, Jacobson A, mRNA poly(A) tail, a 3' enhancer of translational initiation. Mol Cell Biol. 1990, 10(7): 3441–3455.

polysomes / polyribosomes
ribosomes
RNApol
transcription start

>hba2
TTTGAATAAAGTCTGAGTGGGCAGCAGCCTGTGTGTGTGCC
>hba1
TTGAATAAAGTCGGAGTGGGCAGCAGCCTGTGTGTGTGCC
>nfkb1
ATAATAAAGGAAAAGCAAATTGTATGACCTCACCTTGTTTGTCTGTGCA
polyA signal 15 – 30 nt cleavage site GT-rich sequence
spacer

polyA tail: polyadenylation signals
Fitzgerald M, Shenk T, The sequence 5’-AAUAAA-3’ forms part of the recognition site for polyadenylation of late SV40 mRNAs. Cell, 1981, 24:257


Sheets MD, Wickens M, Two phases in the addition of a poly(A) tail. Genes & Development, 1989, 3:1402.

Sheets MD, Wickens M, Two phases in the addition of a poly(A) tail. Genes & Development, 1989, 3:1402.

polyA tail: polyadenylation mechanism

cleavage stimulation factor
cleavage- and polyadenylation-specific factor


RNA transcription: termination in eukaryotes
torpedo model
disengagement / conformational regulation

polyA tail
AAAAAAAAAAAAA
7mG
5’-cap

AAAAAAAAAAAAA
3’-exonuclease
7mG
5’-exonuclease

AAAAAAAAAAAAA
PAB PAB
3’-exonuclease
7mG
5’-exonuclease

AAAAAAAAAAAAA
PAB PAB
eIF4E 7mG

AAAAAAAAAAAAA
PAB PAB
eIF4G
eIF4E 7mG
eIF4A

AAAAAAAAAAAAA
PAB PAB
eIF4G
eIF3
eIF2
Met
eIF4E 7mG
eIF4A
43 S pre-initiation
complex
ribosome

ribosome
mRNA

BIO353 Lecture9 (Transcription SHF 2021 Long

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BIO353 – lecture 9: Transcription – the primary process

the central dogma in Molecular Biology

BIO353.01 – Molecular Genetics shf – 2022

liver cell brain cell

BIO353.01 – Molecular Genetics shf – 2022

TReg cells naïve T cells

BIO353.01 – Molecular Genetics shf – 2022

Brain Heart Kidney Liver Testes

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

The same gene can be transcribed simultaneously multiple times.

Transcription end Transcription end

BIO353.01 – Molecular Genetics shf – 2022

transcription start site

BIO353.01 – Molecular Genetics shf – 2022

Thermus aquaticus Lobster claw

BIO353.01 – Molecular Genetics shf – 2022

Thermus aquaticus Saccharomyces cerevisiae

BIO353.01 – Molecular Genetics shf – 2022

Thermus aquaticus Saccharomyces cerevisiae

BIO353.01 – Molecular Genetics shf – 2022

everything rRNAs mRNAs tRNAs

BIO353.01 – Molecular Genetics shf – 2022

recognition of the transcription stop

BIO353.01 – Molecular Genetics shf – 2022

recognition of the transcription stop

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

RNA pol core enzyme

RNA pol holoenzyme

BIO353.01 – Molecular Genetics shf – 2022

RNA pol holoenzyme

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

closed complex open complex

binding of RNA pol RNA pol melts

BIO353.01 – Molecular Genetics shf – 2022

closed complex intermediate step

BIO353.01 – Molecular Genetics shf – 2022

closed complex open complex

BIO353.01 – Molecular Genetics shf – 2022

s bound to promoter open complex

BIO353.01 – Molecular Genetics shf – 2022

BIO353.01 – Molecular Genetics shf – 2022

• What is the size of the transcription bubble ?

• How can it be determined experimentally ?

RNA pol melts

BIO353.01 – Molecular Genetics shf – 2022

Tw: twist (# of times one strand wraps around the other)

BIO353.01 – Molecular Genetics shf – 2022

closed complex open complex

binding of RNA pol melts DNA adopts