Biochemistry

3382A
Ilka Heinemann, PhD
ilka.heinemann@uwo.ca
MSB 358
Oce hours: Fridays
1 PM- 2 PM

Bacterial transcripIon

TRANSCRIPTION AND
THE REGULATION OF
GENE EXPRESSION

Now that we have all this useful
informa2on, it would be nice to do
something with it.
From the Unix
Programmer s Manual




The RoseOa Stone, inscribed in
196 B.C.

Direct translation of Egyptian to Greek

ESSENTIAL QUESTION

How are the genes of prokaryotes (and

eukaryotes) transcribed to form RNA products
that can be translated into proteins?

All Cells Contain Three Major Classes of RNA

mRNA, rRNA, and tRNA
All three forms parIcipate in protein
synthesis.
All RNAs are synthesized from DNA templates
by DNA-dependent RNA polymerases.
This process is called transcripCon.
Only mRNAs direct the synthesis of proteins.
rRNA, and tRNA are required for translaIon
TranscripIon is Ightly regulated in all cells.

Transcription
DNA-directed synthesis of RNA
TYPE of RNA

FUNCTION

Ribosomal RNA (rRNA)

Components of ribosome

Transfer RNA (tRNA)

Delivery of amino acids

Messenger RNA (mRNA)

Template for protein synthesis

Short nuclear RNAs (snRNAs)

Components of spliceosome

Short interfering RNAs (siRNAs) or

micro RNAs (miRNAs)

Regulation of gene expression

Short nucleolar RNAs (snoRNAs)

Methylation of rRNAs

All human RNAs are transcribed from DNA

templates Not necessarily true for viruses (ex. HIV, HepB) --> some viruses have RNA genes/used as templates

All Cells Contain Three Major Classes of RNA

mRNA, rRNA, and tRNA
Only 3% of genes in a typical cell are
undergoing transcripIon at any given
moment.
The metabolic condiIons and growth status of
the cell dictate which gene products are
needed at any moment.
Only need certain things at certain times --> tightly regulated

HOW ARE GENES TRANSCRIBED IN

PROKARYOTES?
RNA chain grows 5'-3' during transcripIon.
Subsequent hydrolysis of PPi to inorganic
phosphate by pyrophosphatases makes the
polymerase reacIon thermodynamically
favourable.
Energy for chain growth comes from cleaving of diphosphate groups off nucleotides

HOW ARE GENES TRANSCRIBED IN

PROKARYOTES?
In prokaryotes, virtually all RNA is synthesized by a
humans have ~20
single species of DNA-dependent RNA polymerase.
RNA polyms
RNA polymerases link NTPs (ATP, GTP, CTP, and UTP)
in the order specied by base pairing with a DNA
template.
The polymerase moves along the DNA strand in the
3'-5' direcIon. RNA strand is made 5'-3'

TranscripIon
TranscripIon can be divided into four stages:
1) Binding of RNA polymerase
holoenzyme to template
DNA at promoter sites
2) IniCaCon of polymerizaIon
3) Chain elongaCon
4) Chain terminaCon

ConvenIons Used in Expressing the Sequences

of Nucleic Acids and Proteins

ConvenIons Used in Expressing the Sequences

of Nucleic Acids and Proteins

The strand of duplex

DNA that is read by RNA
polymerase is termed
the template stand.

ConvenIons Used in Expressing the Sequences

of Nucleic Acids and Proteins

The strand not read is

the non-template
strand.
Sequence of RNA transcript is parallel to non-template
strand

ConvenIons Used in Expressing the Sequences

of Nucleic Acids and Proteins
All coding strands start with ATG --> parallel to AUG in RNA transcript

The template is read by the RNA

polymerase moving 3'-5' along the
template, so the RNA product, the
transcript, grows in the 5'-3' direcIon.

ConvenIons Used in Expressing the Sequences

of Nucleic Acids and Proteins

By conven2on, when the order of

nucleo2des in DNA is shown as a
single strand, it is the 5'-3' sequence of
nucleo2des in the non-template strand
that is shown. Because it's parallel to RNA, and can be
read directly and can tell what proteins it
codes for relatively easily (instead of
having to read template strand, find
complements, etc)

TranscripIon
TranscripIon can be divided into four stages:
1) Binding of RNA polymerase
holoenzyme to template
DNA at promoter sites

How does the polymerase know where to

start???
Special subunit composiIon: 2'.
Binding of the (Sigma) subunit allows the
polymerase to recognize dierent DNA
sequences that act as promoters.
Promoters are nucleoIde sequences that idenIfy
the locaIon of transcripCon start sites, where
transcripIon begins.
Without bound, the core polymerase can
transcribe DNA into RNA but cannot iniIate
transcripIon.

1. Binding of Polymerase to Template

DNA
Polymerase binds non-specically to DNA with low
anity and migrates along it, looking for promoter.
Sigma subunit recognizes the promoter sequence.
RNA polymerase holoenzyme and promoter form a
closed promoter complex (in which the DNA is not
unwound): Kd = 10-6 to 10-9 M.
Polymerase then unwinds about 12 pairs to form
"open promoter complex : Kd = 10-14 M.
RNA polymerase binding protects a nucleo2de
sequence spanning the region from -70 to +20, where
+1 is dened as the transcrip)on start site.

ProperIes of ProkaryoIc Promoters

Promoters recognized by the factor typically consist
of a 40 bp region on the 5(upstream)
'-side of the transcripIon
start site.
Within the promoter are two consensus sequence
elements:

The Pribnow box near -10, with consensus TATAAT. This

region is ideal for unwinding. Why? (It is rich in As and Ts,
which only form two H bonds per base pair).
The -35 region, with consensus TTGACA: sigma subunit
appears to bind here.

The more the -35 region sequence corresponds to the

consensus sequence of the subunit, the greater is the
eciency of gene transcripIon.

Consensus Sequences for the -35 Region and the

Pribnow Box

Consensus sequences for the -35 region, the Pribnow box, and the initiation site are
shown at the bottom. The numbers represent the percent occurrence of the indicated base.
In this figure, sequences are aligned relative to the Pribnow box.

Bacterial RNA Polymerase (RNAP)

Synthesizes all RNA except primers for replication
4 diff proteins to form 1
polymerase

Core enzyme: 2'

(alpha2, ------------sigma, ------------sigma, omega)
beta

beta'

Positively-charged
cleft binds DNA

Thermus thermophilus RNAP

DNA
template

Mg2+ in active site

DNA non-template

RNA

Sigma factors
Escherichia coli: 7 sigma factors
Bacillus sub2lis: 20 sigma factors
Usually one household sigma factor
Rest are specialized sigma factors, e.g. E. coli 24
(RpoE) - the extracytoplasmic/extreme heat stress
on sigma 24 --> expression of sigma 24 will turn on all genes necessary to respond
sigma facto Turn
to extreme heat

 factor recognizes promoters

RNAP core enzyme

RNAP holoenzyme

is extended and interacts with -35 and -10

regions Rainbow coloured coils stretching across entire DNA section along enzyme

TranscripIon
TranscripIon can be divided into four stages:
1) Binding of RNA polymerase
holoenzyme to template
DNA at promoter sites
2) IniCaCon of polymerizaIon
3) Chain elongaCon

IniIaIon of PolymerizaIon
RNA polymerase has two binding sites for NTPs.
The iniCaCon site prefers to bind ATP and GTP (most
RNAs begin with a purine at 5'-end).
The elongaCon site binds the second incoming NTP.
3'-OH of rst aOacks -P of second to form a new
phosphoester bond (eliminaIng Ppi)
When 6-10 unit oligonucleoIde has been made,
sigma subunit dissociates, compleIng "iniIaIon.

Chain ElongaIon
The core polymerase (without ) is the
elongaIon enzyme.
RNA polymerase is accurate: only about 1 error in 10
000 bases.
Even this error rate is okay, since many transcripts are
made from each gene.
ElongaIon 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.
*G-C base pairs share 3 H bonds, whereas
A-T base pairs, with 2 H bonds, are less stable.

ProkaryoIc IniIaIon and ElongaIon

ProkaryoIc IniIaIon and ElongaIon

DNA FootprinIng: IdenIfying the NucleoIde

Sequence in DNA Where a Protein Binds
DNA footprinCng is a widely used technique to idenIfy
the nucleoIde sequence within DNA where a specic
protein binds (such as a promoter sequence bound to
RNA polymerase holoenzyme).
The protein is incubated with a labelled DNA fragment
containing the sequence where the protein is thought
to bind.
DigesIon with DNase cleaves the DNA backbone in
exposed regions, but not where the DNA-binding
protein is bound.
Analysis of the DNase digests reveals the locaIon of
the protein-binding site on the DNA.

DNA FootprinIng

Supercoiling versus TranscripIon

(a) If the RNA polymerase followed the template strand around the axis of the
DNA duplex, no supercoiling of the DNA would occur but the RNA chain
would be wrapped around the double helix once every 10 bp. This possibility
seems unlikely because it would be difficult to untangle the transcript from
the DNA duplex.
(b)Alternatively, gyrases and topoisomerases could remove the torsional stresses
induced by transcription.

TranscripIon
TranscripIon can be divided into four stages:
1) Binding of RNA polymerase
holoenzyme to template
DNA at promoter sites
2) IniCaCon of polymerizaIon
3) Chain elongaCon
4) Chain terminaCon

Chain TerminaIon
Two types of transcripIon terminaIon
mechanisms operate in bacteria: One depends
on Rho terminaCon factor
Rho is an ATP-dependent helicase.
It moves along RNA transcript, nds the
transcripIon bubble, unwinds the DNA:RNA
hybrid, and releases RNA chain.

It is likely that the RNA polymerase stalls in a

G:C-rich terminaIon region, allowing rho
factor to overtake it.

TranscripIon TerminaIon by rho Factor

Model of Rho-mediated termination

Rho recognizes rut site, then

closes around RNA and
advances toward RNAP
Rho causes allosteric change
in RNAP that releases
template
DJ Koslover et al. (2012) J Mol Biol 423:664-76

Intrinsic TerminaIon
The second terminaIon mechanism is termed intrinsic
terminaCon.
Here, terminaIon is determined by specic sequences
in the DNA called terminaCon sites.
TerminaIon sites consist of three structural features:
Inverted repeats, rich in G:C, which form a stable stem-
loop structure in RNA transcript
A non-repeaIng segment that punctuates the inverted
repeats
A run of 6-8 As in the DNA template, coding for Us in the
transcript

The TerminaIon Site for

E. coli trp Operon

Operons
Operon (in bacteria): two or more genes
transcribed together as one RNA molecule
The mRNA is polycistronic
Genes in an operon usually have related
functions

Overview of bacterial transcription

dissociates

Fig 12-3 from Watson et al., Molecular

Biology of the Gene, 6th ed.

Summary of bacterial transcription

Transcription of genes is carried out by RNA
polymerase (RNAP), a multisubunit complex
Bacterial RNAP recognizes promoters with a
defined consensus sequence via the subunit
DNA strands are separated and RNA synthesis
occurs, possibly after abortive initiation
Termination is mediated by a termination hairpin
structure or by Rho factor