Gene Expression
Prokaryotic Gene Transcription
G & G Chapter 29
9/14/11
Thomas Ryan, Ph.D.
Biochemistry and Molecular Genetics
tryan@uab.edu
Modified Central Dogma of Francis Crick (1958)
Prokaryotic Chromosome (E. coli)
• Large circular chromosome 4.6 x 106 bp
• Genome forms a compact
structure called the nucleoid
• DNA organized in 50-100 loops
(domains)
• The ends of loops are
constrained by binding to
protein structure which is in
contact with cell membrane
Additional Forms of RNA
All cells transcribe 3 major types of RNA molecules:
messenger RNA (mRNA)
ribosomal RNA (rRNA)
transfer RNA (tRNA)
In archea and eubacteria:
3 RNAs are produced by a single
DNA dependent RNA polymerase
In eukaryotes:
3 RNAs are produced by 3 distinct
DNA dependent RNA polymerases (I, II, & III)
RNA Quantity and Stability
rRNA & tRNA (stable):
Not degraded rapidly (although extensively processed)
 Rapidly growly E. coli:
 80% of RNA is rRNA
 15% is tRNA
 Ribosome number [rRNA] is proportional to growth rate

mRNA (translated):
~2 to 5% of total RNA is mRNA
 unstable with a t1/2 ≈ 2-3 min - allows regulation at the
level of mRNA synthesis

mRNA Degradation by RNases
Exonucleases (3’—> 5’ only in bacteria)
5’
3’
Endonucleases (internal cuts)
5’
3’
Transcription: The Players
Ribonucleotides (NTPs)
Template (DNA)
DNA Dependent RNA Polymerase
Transcription factors
Transcription: DNA to mRNA
G & G page 907
Major bases found in DNA and RNA
DNA
Adenine
Cytosine
Guanine
Thymine
thymine-adenine base pair
RNA
Adenine
Cytosine
Guanine
Uracil (U)
uracil-adenine base pair
DNA Dependent RNA Polymerase: Catalysis Reaction
Growing RNA
strand

Direction of synthesis
is 5’ to 3’

No primer required

Template required
Template
strand DNA
A
C
n
T
G
n+1
A
T
C
G
Chromosome is divided into genes, which encode RNA and
protein products.
Gene
DNA 5’
3’
[
AGTC
TCAG
]
3’
5’
Transcription
RNA
AGUC
5’
Chemical differences
ribose vs. deoxyribose
*uracil vs. thymine
*


3’
single stranded
complementary to one strand
of DNA (bottom in this case)
Naming the DNA Strands of a Gene
nontemplate
nontranscribed
top
5’
3’
template
transcribed
[
bottom
5’
AGTC
TCAG
]
3’
DNA
5’
Transcription
AGUC
3’
RNA
Both DNA Strands Encode Genes
And Can Be Transcribed.
3’
RNA # 2
5’
Transcription
[
[
]
Transcription
5’
3’
RNA # 1
Gene # 2
P
Gene # 1
]
P
5’
3’
3’
DNA
5’
Prokaryotic RNA polymerase
•
Synthesizes all major classes of RNA
 messenger RNA (mRNA)
 ribosomal RNA (rRNA)
 transfer RNA (tRNA)
• Multisubunit Protein
• Holoenzyme = a2bb' catalyzes initiation of RNA
synthesis specifically at a promoter
• Core enzyme= a2bb' catalyzes elongation of the
RNA chain
Transcription in Prokaryotes
Only a single RNA polymerase
 In E.coli, RNA polymerase is 465 kD complex,
with 2 a, 1 b, 1 b', 1 
 a subunits appear to be essential for assembly and
for activation of enzyme by regulatory proteins
 b binds NTPs, interacts with , and forms catalytic
site with b'
 b' binds nonspecifically to DNA and forms
catalytic site with b
  recognizes promoter sequences on DNA, aids in
melting the dsDNA by binding nontemplate strand
Prokaryotic Transcription Cycle
 Initiation
 Holoenzyme binds to the promoter, unwinds DNA, and
forms phosphodiester bonds between 7 to 12 nucleotides
 Need  to recognize promoter
 Elongation
  dissociates
 Core enzyme elongates RNA with high processivity
 Termination
 Polymerase dissociates from template DNA and releases
new RNA
 Rho()-factor dependent or independent.
Binding of RNA Polymerase to Template DNA
• Polymerase binds nonspecifically to DNA with low
affinity and migrates, looking for promoter
• Sigma () subunit recognizes promoter sequence
• RNA polymerase holoenzyme and promoter form
"closed promoter complex" (DNA not unwound) - Kd =
10-6 to 10-9 M
• Polymerase unwinds about 14 base pairs of DNA to
form "open promoter complex" - Kd = 10-14 M
RNA Polymerase Binding to DNA - Promoter Search
Nonspecific binding to DNA:
(i.e., to non-promoter DNA)
holo - Ka ≈ 107/M
(very rough numbers)
Specific binding to DNA:
(i.e., to promoter)
holo - Ka ≈ 1014/M
(actual value depends on promoter!)
Note: in E. coli there are:
~3000 molecules of RNAP core
~1000 molecules of 
~1000 promoters
virtually unlimited nonspecific DNA sites

Holoenzyme searches for promoters by sliding along DNA and by
intramolecular transfer on the chromosome.
Properties of Promoters
See Figure 29.3
• Promoters typically consist of 40 bp region on the
5'-side of the transcription start site
• Two consensus sequence elements:
• The "-35 region", with consensus TTGACA
• The Pribnow box near -10, with consensus
TATAAT - this region is ideal for unwinding - why?
Prokaryotic Promoters
G & G Fig. 29.3
Consensus  Factor Promoters
Stages of Transcription
•
•
•
•
See G & G Figure 29.2
binding of RNA polymerase holoenzyme
at promoter sites
initiation of polymerization
chain elongation
chain termination
Transcriptional Events
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 PPi)
• When 7-12 unit oligonucleotide has been made, sigma
subunit dissociates, completing "initiation"
• Note mode of action of rifamycin (rifampicin)--binds to b
subunit of RNA polymerase and blocks first phosphodiester
bond. Specific for prokaryotic RNA polymerase!
Events at
initiation of
transcription
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
The Elongation Complex
• RNAP core enzyme covers about 60 bp of DNA, with
about 17 bp unwound = transcription bubble.
• The bubble must contact the active site for
polymerization.
• At the beginning of the bubble, the DNA is unwound,
implicating a helicase activity.
• At the end of the bubble, the DNA is rewound.
Supercoiling Versus Transcription
G & G Fig. 29.4
Inhibitors of Transcription
Intercalates G:C basepairs
b subunit of RNAP
Transcription Termination
Two mechanisms
• Rho - the termination factor protein
– rho is an ATP-dependent helicase
– it moves along RNA transcript, finds the
"bubble", unwinds it and releases RNA chain
• Specific sequences - termination sites in DNA
– inverted repeat, rich in G:C, which forms a stemloop in RNA transcript
– 6-8 As in DNA coding for Us in transcript
– “Intrinsic Termination”
Transcription Termination
Sequence Dependent / Factor Independent
(Intrinsic Termination)
Stem Loop
Structure
Transcription Termination: Rho-factor Dependent
Regulation of Prokaryotic Gene Transcription
Regulation occurs at every level
Transcription (RNA synthesis)
RNA stability, processing, localization
Translation
Post-translational
Regulation of Prokaryotic Gene Transcription
Introduction
DNA:protein, protein:protein interactions
Organization of genes into operons
lac and trp operons
Positive control or activation
Negative control or repression
Attenuation control of transcription
Transcription Terminology


promoter = DNA site recognized by RNA polymerase for specific
transcriptional initiation
terminator = region of DNA containing signals for
termination of transcription

structural gene = DNA that encodes a protein (or RNA product?)

cistron = gene, mRNA specified by the structural gene

coding region = structural gene or cistron

open reading frame (ORF) = coding region (i.e., no stop codons)

operon = promoter + (gene)n + terminator, where n ≥1
1 transcript ≥ 1 cistron
General Rules for DNA Binding Proteins

3D structure of regulatory proteins - most of them are
homodimers

DNA sequence recognized by homodimers are typically
palindromic (inverted repeats); they have dyad symmetry

Each monomer of the homodimer is in contact with bases in half
of the palindromic sequence

This allows the protein coding region to remain relatively small
while the protein recognizes a large sequence that is quite specific
Transcription factors
DNA binding proteins that decrease (repressors) or increase
(activators) the efficiency of transcription at the promoter.
Transcription is
the primary site
of control in
prokaryotes
Promoters drive
the expression of
genes
Prokaryotic
genes can be
arranged in
operons
Transcription Regulation in Prokaryotes
• Genes encoding for enzymes of metabolic
pathways are grouped in clusters on the
chromosome - called operons
• This allows coordinated regulation and gene
expression
• A regulatory sequence adjacent to such a unit
determines whether it is transcribed - this is the
‘operator’
• Regulatory proteins interact with operators to
control transcription of the genes
General Organization of Operons
Operators can be upstream, downstream, or overlapping with
the promoter.
Regulatory proteins that bind to the operator can influence the
access of RNA polymerase to the promoter thereby affecting
the rate of transcription initiation.
Coordinate Regulation
• Expression of several or numerous genes can be
controlled simultaneously.
• Operon: a set of genes that are transcribed from the
same promoter and controlled by the same operator
site and regulatory proteins.
• Regulon: a set of genes (and/or operons) expressed
from separate promoter sites, but controlled by the
same regulatory molecule. Global regulons may
coordinate expression of many genes and operons, and
may induce some, but repress others.
Global Regulation Via Sigma Factors
Different promoter architectures are recognized by
sigma factor subunit of RNA polymerase
Gene Expression
Prokaryotic Gene Transcription
(Cont.)
&
Eukaryotic Transcription
Histones and Chromatin
G & G Pages 336-340, Chapter 29,
9/14/11
Thomas Ryan, Ph.D.
Biochemistry and Molecular Genetics
tryan@uab.edu