The Mechanism of

Transcription in
Bacteria
FROM DNA TO RNA

Dr. Saadia Naseem

BIO240
COMSATS University Islamabad

BIO240_Introductory Molecular Biology

 Transcription

The synthesis of RNA molecules using DNA

strands as the templates so that the genetic
information can be transferred from DNA to
RNA.
 Transcription
Genes can be expressed with different efficiencies
Similarity between
replication and transcription

◼ Both processes
use DNA as the
template.
◼ Phosphodiester
bonds are formed
in both cases.
◼ Both synthesis
directions are from
5´ to 3´.
The chemical structure of RNA
(A) RNA contains
the sugar ribose,
which differs from
deoxyribose, the
sugar used in
DNA, by the
presence of an
additional –OH
group.
(B) RNA contains
the base uracil,
which differs from
thymine, the
equivalent base in
DNA, by the
absence of a –
CH3 group
 Uracil forms base pairs with adenine
RNA can fold into specific structures.

(A) Diagram of a folded RNA structure showing only conventional base-pair interactions. (B)
Structure with both conventional (red) and nonconventional (green) base-pair interactions. (C)
Structure of an actual RNA, one that catalyzes its own splicing. Each conventional base-pair
interaction is indicated by a “rung” in the double helix. Bases in other configurations are
indicated by broken rungs.
DNA is transcribed by the enzyme RNA polymerase
The RNA polymerase (pale blue) moves
stepwise along the DNA, unwinding the DNA
helix at its active site indicated by the Mg2+
(red), which is required for catalysis. As it
progresses, the polymerase adds nucleotides
one by one to the RNA chain at the
polymerization site, using an exposed DNA
strand as a template. The RNA transcript is
thus a complementary copy of one of the two
DNA strands. A short region of DNA/RNA helix
(approximately nine nucleotide pairs in length)
is formed only transiently, and a “window” of
DNA/RNA helix therefore moves along the
DNA with the polymerase as the DNA double
helix reforms behind it. The incoming
nucleotides are in the form of ribonucleoside
triphosphates (ATP, UTP, CTP, and GTP), and
the energy stored in their phosphate–
phosphate bonds provides the driving force
for the polymerization reaction.
Differences between
replication and transcription

replication transcription

template double strands single strand

substrate dNTP NTP

primer yes no

Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pair A-T, G-C A-U, T-A, G-C

5' GCAGTACATGTC 3' coding
strand
3' CGTCATGTACAG 5' template
strand

transcription

5' GCAGUACAUGUC 3' RNA

Principal Types of RNAs Produced in
Cells
 RNA Polymerase Structure

RNA polymerase from E. coli consists of five subunits

◼ 2 very large subunits are b (150 kD) and b’ (160 kD)
◼ Sigma (s) at 70 kD
◼ Alpha (a) at 40 kD – 2 copies present in holoenzyme
◼ Omega (w) at 10 kD
◼ Not required for cell viability or in vivo enzyme activity
◼ Appears to play a role in enzyme assembly
RNA Polymerase Structure
b
b s
a
a

subunit MW function
Determine the DNA to be
a 36512
transcribed

b 150618 Catalyze polymerization

b 155613 Bind & open DNA template

Recognize the promoter

s 70263
for synthesis initiation
 Sigma as a Specificity Factor
◼ Core enzyme without the s subunit could not transcribe
viral DNA, yet had no problems with highly nicked calf
thymus DNA
◼ With s subunit, the holoenzyme worked equally well on
both types of DNA

Introductory Mol. Biol-CIIT

Promoters
◼ Nicks and gaps are good sites for RNA
polymerase to bind nonspecifically
◼ Presence of the s-subunit permitted
recognition of authentic RNA polymerase
binding sites
◼ Polymerase binding sites are called
promoters
◼ Transcription that begins at promoters is
specific, directed by the s-subunit

Introductory Mol. Biol-CIIT

Binding of RNA Polymerase to
Promoters
◼ How tightly does core
enzyme v. holoenzyme bind
DNA?
◼ Experiment measures
binding of DNA to enzyme
using nitrocellulose filters
◼ Holoenzyme binds filters
tightly
◼ Core enzyme binding is
more transient

Introductory Mol. Biol-CIIT

 Polymerase/Promoter Binding
◼ Holoenzyme binds DNA
loosely at first
◼ Complex loosely bound at
promoter = closed promoter
complex, dsDNA in closed
form
◼ Holoenzyme melts DNA at
promoter forming open
promoter complex -
polymerase tightly bound
Core Promoter Elements
◼ There is a region common to bacterial promoters described as
6-7 bp centered about 10 bp upstream of the start of
transcription = -10 box
◼ Another short sequence centered 35 bp upstream is known as
the -35 box
◼ Comparison of thousands of promoters has produced a
consensus sequence for each of these boxes
 Promoter Sequences

◼ Consensus sequences:
◼ -10 box sequence approximates TAtAaT
◼ -35 box sequence approximates TTGACa
Signals Encoded in DNA Tell RNA
Polymerase Where to Start and Stop
UP Element
◼ UP element is a promoter, stimulating
transcription by a factor of 30
◼ UP is associated with 3 “Fis” sites which are
binding sites for transcription-activator protein
Fis, not for the polymerase itself
The rrnB P1 Promoter
Transcription Initiation
◼ Transcription initiation was assumed to end
as RNA polymerase formed 1st
phosphodiester bond
◼ Carpousis and Gralla found that very small
oligonucleotides (2-6 nt long) are made
without RNA polymerase leaving the DNA
◼ Abortive transcripts such as these have been
found up to 10 nt
 Stages of Transcription Initiation
◼ Formation of a closed
promoter complex
◼ Conversion of the closed
promoter complex to an open
promoter complex
◼ Polymerizing the early
nucleotides – polymerase at
the promoter
◼ Promoter clearance –
transcript becomes long
enough to form a stable
hybrid with template
The Functions of s
◼ Gene selection for transcription by s causes
tight binding between RNA polymerase and
promoters
◼ Tight binding depends on local melting of
DNA that permits open promoter complex
◼ Dissociation of s from core after sponsoring
polymerase-promoter binding
 Reuse of s

◼ During initiation s can be recycled for additional use in a

process called the s cycle
◼ Core enzyme can release s which then associates with
another core enzyme
Sigma May Not Dissociate from
Core During Elongation

◼ The s-factor changes its relationship to the

core polymerase during elongation
◼ It may not dissociate from the core
◼ May actually shift position and become more
loosely bound to core
Local DNA Melting at the
Promoter
◼ size of the DNA transcription bubble in
complexes where transcription was active
was found to be 17-18 bp
Structure and Function of s
◼ Genes encoding a variety of s-factors have
been cloned and sequenced
◼ There are striking similarities in amino acid
sequence clustered in 4 regions
◼ Conservation of sequence in these regions
suggests important function
◼ All of the 4 sequences are involved in binding
to core and DNA
Homologous Regions in Bacterial
s Factors

Introductory Mol. Biol-CIIT

E. coli s70
◼ Four regions of high
sequence similarity
are indicated
◼ Specific areas that
recognize the core
promoter elements, -
10 box and –35 box
are notes
Region 1

◼ Role of region 1 appears to be in preventing

s from binding to DNA by itself
◼ This is important as s binding to promoters
could inhibit holoenzyme binding and
thereby inhibit transcription
Region 2
◼ This region is the most highly conserved of
the four
◼ There are four subregions – 2.1 to 2.4
◼ 2.4 recognizes the promoter’s -10 box
◼ The 2.4 region appears to be a-helix
Regions 3 and 4
◼ Region 3 is involved in both core and DNA
binding
◼ Region 4 is divided into 2 subregions
◼ This region seems to have a key role in
promoter recognition
◼ Subregion 4.2 contains a helix-turn-helix
DNA-binding domain and appears to govern
binding to the -35 box of the promoter
Summary
◼ Comparison of different s gene sequences reveals 4
regions of similarity among a wide variety of sources
◼ Subregions 2.4 and 4.2 are involved in promoter -10
box and -35 box recognition
◼ The s-factor by itself cannot bind to DNA, but DNA
interaction with core unmasks a DNA-binding region
of s
◼ Region between amino acids 262 and 309 of b’
stimulates s binding to the nontemplate strand in the
-10 region of the promoter
 Role of a-Subunit in UP Element
Recognition
◼ RNA polymerase itself can recognize an
upstream promoter element, UP element
◼ While s-factor recognizes the core promoter
elements, what recognizes the UP element?
◼ It appears to be the a-subunit of the core
polymerase
 Role of a-Subunit in UP Element
Recognition
◼ RNA polymerase binds to a core
promoter via its s-factor, no help
from C-terminal domain of a-
subunit
◼ Binds to a promoter with an UP
element using s plus the a-
subunit C-terminal domains
◼ Results in very strong interaction
between polymerase and
promoter
◼ This produces a high level of
transcription
Elongation
◼ After transcription initiation is accomplished,
core polymerase continues to elongate the
RNA
◼ Nucleotides are added sequentially, one
after another in the process of elongation
Function of the Core Polymerase
◼ Core polymerase contains the RNA
synthesizing machinery
◼ Phosphodiester bond formation involves the
b- and b’-subunits
◼ These subunits also participate in DNA
binding
◼ Assembly of the core polymerase is a major
role of the a-subunit
 Role of b in Phosphodiester Bond
Formation
◼ Core subunit b lies near the active site of the
RNA polymerase
◼ This active site is where the phosphodiester
bonds are formed linking the nucleotides
◼ The s-factor may also be near nucleotide-
binding site during initiation phase
 Role of b’ and b in DNA Binding
◼ In 1996, Evgeny Nudler and colleagues showed
that both the b- and b’-subunits are involved in
DNA binding
◼ They also showed that 2 DNA binding sites are
present
◼ A relatively weak upstream site
◼ DNA melting occurs
◼ Electrostatic forces are predominant
◼ Strong, downstream binding site where
hydrophobic forces bind DNA and protein
together
RNA-DNA Hybrid
◼ The area of RNA-DNA hybridization within
the E. coli elongation complex extends from
position –1 to –8 or –9 relative to the 3’ end
of the nascent RNA
◼ In T7 the similar hybrid appears to be 8 bp
long
Topology of Elongation
◼ Elongation of transcription involves polymerization of
nucleotides as the RNA polymerase travels along the
template DNA
◼ Polymerase maintains a short melted region of
template DNA
◼ DNA must unwind ahead of the advancing
polymerase and close up behind it
◼ Strain introduced into the template DNA is relaxed by
topoisomerases
Termination of Transcription
◼ When the polymerase reaches a terminator at
the end of a gene it falls off the template and
releases the RNA
◼ There are 2 main types of terminators
◼ Intrinsic terminators function with the RNA
polymerase by itself without help from other
proteins
◼ Other type depends on auxiliary factor called r
(rho), these are r-dependent terminators
Rho-Independent Termination
◼ Intrinsic or r-independent termination
depends on terminators of 2 elements:
◼ Inverted repeat followed immediately by
◼ T-rich region in nontemplate strand of the
gene
◼ An inverted repeat predisposes a transcript to
form a hairpin structure
Inverted Repeats and Hairpins
◼ The repeat at right is
symmetrical around its
center shown with a dot
◼ A transcript of this
sequence is self-
complementary
◼ Bases can pair up to
form a hairpin as seen
in the lower panel
 Model of Intrinsic Termination
Bacterial terminators act by:
◼ Base-pairing of something to
the transcript to destabilize
RNA-DNA hybrid
◼ Causes hairpin to form
◼ Causing the transcription to
pause
◼ Causes a string of U’s to be
incorporated just downstream
of hairpin
Rho-Dependent Termination
◼ Rho caused depression of the ability of RNA
polymerase to transcribe

◼ This depression is due to termination of

transcription

◼ After termination, polymerase must reinitiate

to begin transcribing again
 Rho Causes Production of Shorter
Transcripts
◼ Synthesis of much smaller RNAs occurs in the
presence of r compared to those made in the
absence

◼ loss of transcript size is not because of RNase

activity BUT
◼ Because of r factor
Rho serves to Releases the RNA
Transcripts from the DNA Template
 Mechanism of Rho

◼ No string of T’s in the r-

dependent terminator, just
inverted repeat to hairpin
◼ Binding to the growing
transcript, r follows the RNA
polymerase
◼ It catches the polymerase as
it pauses at the hairpin
◼ Releases transcript from the
DNA-polymerase complex
by unwinding the RNA-DNA
hybrid