Lecture 8: Bacterial Transcription

Part A: Genes and Transcripts

How do bacteria transcrib

certain DNA genes into RNA

How do bacteria control which genes the

transcribe, and how much RNA they make?

Lecture 08A: Genes and Transcripts MCB102 Spring 2022 1

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 RNA synthesis

NTP selected by base pairin

to template DNA strand

Always 5’→3’ on growing RNA strand

Nucleophilic attack
by 3’ O RNA synthesis very simila
on the α phosphat to DNA synthesis
of a new NTP

Lecture 08A: Genes and Transcripts MCB102 Spring 2022 2

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 E coli RNA polymerase

5 core subunits: ααββ’ω

Synthesizes all RNAs — mRNA, rRNA, tRNA, other non-coding RNAs
Core RNA Pol can synthesize RN
but cannot recognize promoters
Active site spli
between β and β’

No proofreading 3’→5’ exonucleas

Error rate 10-4 to 10-5
Lecture 08A: Genes and Transcripts MCB102 Spring 2022 3
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 Transcription

50 - 90 nucleotides / second

~17 base pair

unwoun
bubble

~8 base pair
nascent RNA an
DNA template

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 Transcription and topology

(Wr ↓) (Wr ↑)

Retwisting (Tw ↑) Untwisting (Tw ↓)

No change in Lk = Tw + Wr No change in Lk = Tw + Wr

Lecture 08A: Genes and Transcripts MCB102 Spring 2022 5

 RNA synthesis

Only one strand of DNA duplex is transcribed into RNA

The template strand is used by RNA polymerase
The RNA transcript sequence matches the nontemplate stran
RNA and non-template-strand DNA both complementary to template strand

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 Template and product sequences

Only one strand of DNA duplex is transcribed into RNA

The template strand is used by RNA polymerase
The RNA transcript sequence matches the nontemplate stran
RNA and non-template-strand DNA both complementary to template strand

complementary

same sequenc
(except Ts and Us)

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 Transcripts and promoters

Transcription start
only at speci c site
DNA sequence matching RN
called promoters
Non-template stran
Coding strand
RNA transcript
Arrow points 5’→3’
5’ 3’
3’ 5’

Template stran
Reverse complement to RNA

Lecture 08A: Genes and Transcripts MCB102 Spring 2022 8

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 Gene organization

Map of all transcripts produced from a 36,000 bp adenovirus genome

Different transcripts on either strand

Some regions not transcribe
on either strand

Some regions included i

transcripts on both strands

Lecture 08A: Genes and Transcripts MCB102 Spring 2022 9

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 Lecture 8: Bacterial Transcription

Part B: DNA-binding Proteins

How do bacteria transcrib

certain DNA genes into RNA

How do bacteria control which genes the

transcribe, and how much RNA they make?

Lecture 08B: DNA-binding Proteins MCB102 Spring 2022 10

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 E coli σ factors

σ facto
Cor (one of several)
ααββ’ω

σ factor recognize
promoter sequence

Lecture 08B: DNA-binding Proteins MCB102 Spring 2022 11

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 E coli promoters

σ70
Coding (non-template
strand sequences

Two speci c
nucleotide sequences
Sequence unimportan
Distance is important
Different σ factor
bind different sequences
First base of the RNA is +
Bases before the RNA are negativ
There is no 0
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 Real E coli promoters

σ70

Various real promoter

very similar but not identica

Higher af nity for σ facto

More RNA Pol bindin
More RNA produced

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 UP elements

α α σ70

Very strong promoter

also have an UP elemen
that binds core polymerase α

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 DNA-binding proteins

6 nt motif 17 nt spacer 6 nt motif

Proteins bin
intact double helix

Interactions with th
10.5 bp major groove of the DNA
1 turn

Contacts lie on the same physica

21 bp
side of the double helix
2 turns
Lecture 08B: DNA-binding Proteins MCB102 Spring 2022 15
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 Major groove contacts

Major groove presents a distinct molecular surface for each base pair
Sequence-speci c protein-DNA interactions

Minor groove less distinc

(and less accessible)

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 Protein-nucleobase interactions

Amino acid side-chains form base-speci c favorable interactions

Details of protein-DNA interaction are different for each protein

les
favorable

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 Lecture 8: Bacterial Transcription
Part C: Transcription Cycle

How do bacteria transcrib

certain DNA genes into RNA

How do bacteria control which genes the

transcribe, and how much RNA they make?

Lecture 08C: Transcription Cycle MCB102 Spring 2022 18

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 Promoter binding

Double helix is closed

Core RNA Pol + σ facto

bind (form closed complex
and unwind (form open complex
as separate steps
Double helix is ope
~12 base pairs

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 Abortive initiation

RNA Polymerase begins RNA synthesi

without a primer
Cycles of abortive initiation
Release short RNA product, start over
Abortive Initiation 8 - 15 nt RNA

8 - 15 nt RNA
Release of σ facto
at promoter clearance

Eventual promoter clearance

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 Transcription elongation

After promoter clearance

RNA polymerase elongatio
is highly processive (~100,000 nt)

No mechanism to re-start RNA synthesi

after polymerase disengagement

Lecture 08C: Transcription Cycle MCB102 Spring 2022 21

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 Intrinsic transcription termination

Intrinsic (ρ independent) terminatio

RNA (and DNA) sequence alone
stop transcription, no other proteins

RNA stem-loop followed by poly-U

poly-U tract in RNA

pauses transcription and weak UA base pairin
with template DNA

RNA stem-loop from RNA-RNA base pairin

May interact with RNA polymerase
May directly pull apart mRNA-DNA duplex?

Lecture 08C: Transcription Cycle MCB102 Spring 2022 22

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 ρ-dependent termination

ρ-dependent terminatio
ATP-dependent helicase ρ required

rut site with transcription pause downstream

ρ helicase loads at rut site in nascent RNA

pause allows ρ to catch up with polymerase

May interact with RNA polymerase

May directly pull apart mRNA-DNA duplex?

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 Lecture 8: Bacterial Transcription

Part D: Transcription Regulation

How do bacteria transcrib

certain DNA genes into RNA

How do bacteria control which genes the

transcribe, and how much RNA they make?

Lecture 08D: Transcription Regulation MCB102 Spring 2022 24

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 Transcriptional repressors

Represso
Decreases transcriptio

e.g. by physically blockin

RNA polymerase

Loss of a represso
or binding site

constitutive
always transcribed
Lecture 08D: Transcription Regulation MCB102 Spring 2022 25
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 Transcriptional activators

Activato
Increases transcriptio

e.g. by favorable interaction

with RNA polymerase

Loss of an activato
or binding site

uninducible
not transcribed
Lecture 08D: Transcription Regulation MCB102 Spring 2022 26
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 Transcriptional regulators

Promoters can have combinations of activator and repressor binding site

Complicated programs of gene expression control

Lecture 08D: Transcription Regulation MCB102 Spring 2022 27

 Lactose metabolism

lacY
Cells express enzymes for lactose metabolis
only when lactose is presen
and when glucose (preferred food) absent

lacZ
Allolactose is a minor product of lac
directly sensed within the cel
as an indicator of lactose

Lecture 08D: Transcription Regulation MCB102 Spring 2022 28

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 The lac operon

Second, weake
lacI binding site

lacI repressor protei

expressed constitutively Binding site for lacI protei

Symmetric / inverted repeat

Lecture 08D: Transcription Regulation MCB102 Spring 2022 29

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 The lacI repressor

Fits in to major groove

Helix-turn-helix DNA-binding motif

One individual lac

binds one AATTGT motif

Lecture 08D: Transcription Regulation MCB102 Spring 2022 30

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 LacI repressor dimers and tetramers

lacI exist
One pair of lacI protein as a dimer of dimers
binds one operator site

Symmetric dimer bind

symmetric operator

Af nity for operator ~10-10

Speci city for operator ~10
Just good enough to bind

Lecture 08D: Transcription Regulation MCB102 Spring 2022 31

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 lac regulation by lactose

lac operon transcribe

when operator unoccupied
Allolactose binding
allosterically reduce
the DNA af nity of lacI

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 lac regulation by glucose

Activator protein CR
binds near lac promoter

CRP interacts with

RNA Pol α subunit

CRP interaction recruits

RNA Pol to promoter
leading to more transcription

Lecture 08D: Transcription Regulation MCB102 Spring 2022 33

 CRP and cyclic AMP

CRP binds DNA only when bound to cyclic AMP (cAMP)

Bacteria make cAMP in response to low glucose

cyclic AMP

Lecture 08D: Transcription Regulation MCB102 Spring 2022 34

 lac regulation

lactose present Multiple regulators interac

in different way

no lactose no expression No general rule / prediction

glucos
low

Lecture 08D: Transcription Regulation MCB102 Spring 2022 35

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 Transcriptional regulators

Activators
Bind speci c DNA sequence
Increase transcriptio
e.g. by interacting with RNA Pol
increasing effective promoter af nity
Remove (by mutation) activator or
binding site: uninducible, cannot turn on

Repressors
Bind speci c DNA sequence
Decrease transcriptio
e.g. by blocking RNA Pol fro
binding the promoter
Remove (by mutation) repressor or
binding site: constitutive, cannot turn off
Lecture 08D: Transcription Regulation MCB102 Spring 2022 36
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