Chapter 5.

Transcription (RNA Synthesis)

Overview
Transcription is the process of making an RNA copy of a
single gene. Genes are specific regions of the DNA on a
chromosome.

The enzyme used in transcription is called DNA-

dependant RNA polymerase or “RNA polymerase”. There
are several forms of RNA polymerase. In eukaryotes, most
genes are transcribed by RNA polymerase 2.

The raw materials for the new RNA are the 4 ribonucleoside
triphosphates: ATP, CTP, GTP, and UTP. It’s the same ATP as is used
for energy in the cell.

As with DNA replication, transcription proceeds 5- to 3’: new bases

are added to the free 3’ OH group. Sequence corresponds to the sense
strand (coding strand).
Cont’d

Unlike replication, transcription does not need to build on a primer.

Instead, transcription starts at a region of DNA called a “promoter”.
For protein-coding genes, the promoter is located a few bases 5’ to
(upstream from) the first base that is transcribed into RNA

Promoter sequences are very similar to each other, but not identical.
If many promoters are compared, a “consensus sequence” can be
derived. All promoters would be similar to this consensus sequence

Promoter is involved in binding of RNA polymerase to initiate

transcription
 The template of RNA synthesis is the antisense strand

It may be called "minus strand”.

The other DNA strand may be termed "non-template strand", "coding

strand", "plus strand", or "sense strand". Since both DNA coding strand
and RNA strand are complementary to the template strand, they have
the same sequences except that T in the DNA coding strand is replaced
by U in the RNA strand.
Transcription starts with RNA polymerase binding to the
promoter.

This binding only occurs under some conditions: when the gene is “on”.
Various other proteins (transcription factors) help RNA polymerase bind to
the promoter. Other DNA sequences further upstream from the promoter
are also involved.

Once it is bound to the promoter, RNA polymerase unwinds a small section

of the DNA and uses it as a template to synthesize an exact RNA copy of
the DNA strand.

The transcription start site (defined as nucleotide +1) is close to the

promoter and upstream of the start (AUG) codon.
Key terms defined in transcription

• Coding strand of DNA has the same sequence as mRNA.

•Downstream identifies sequences proceeding further in the direction of

expression; for example, the coding region is downstream of the
initiation codon.
+1

Gene X
upstream
downstream

Primary transcript

m7Gppp AAAAAn mRNA

Upstream identifies sequences proceeding in the opposite direction from
expression; for example, the bacterial promoter is upstream from the
transcription unit, the initiation codon is upstream of the coding region.

Transcription unit is the distance between sites of initiation and

termination by RNA polymerase; may include more than one gene.
- Each protein coding gene is an independent transcription unit, and the
strand that is transcribed can differ among genes.

+1
Promoter Terminator
DNA Transcribed region Sense strand
Antisense strand
Transcription
RNA
Structure of a typical transcription unit
Cistron – is a structural gene (a coding sequence of DNA) encoding a
single polypeptide chain.

Open reading frame (ORF) is any sequence of bases (in DNA or RNA)
that could encode a protein.

RNA Terminator is a sequence of DNA, represented at the end of the

transcript, that causes RNA polymerase to terminate transcription.

Primary transcript is the original unmodified RNA product corresponding

to a transcription unit.
5.1. Transcription in Prokaryotes

►Main Classes of RNA

1) rRNA (ribosomal) (80%)
• 18S, 28S and 5S rRNA
• structural and functional components of ribosomes
• highly abundant and stable
2) mRNA (messenger)(<5%)
• typically about 3-500 bases long
• encode proteins
• multiple types, usually not abundant, unstable
3) tRNA (transfer) (15%)
• very small - about 100 bases long
• key role in translation
RNA polymerase has many functions

• Scan DNA and identify promoters

• Bind to promoters
• Initiate transcription
• Elongate the RNA chain
• Terminate transcription
• Be responsive to regulatory proteins (activators and
repressors)

RNAP is a multisubunit enzyme

RNA Polymerase

Subunits Number Mass(KD)

• Prokaryotes contain a single
type of RNAP with five
subunits- forming a 465 kD
α 2 37
complex called Holoenzyme.
• All three classes of RNAs are β 1 151
transcribed by the same RNA
polymerase (RNAP for short). β' 1 155

σ 1 70
omega 1 11
(ώ)
Core enzyme is 2 , 1 , 1 ’ (can transcribe but it can’t find
promoters).
Holoenzyme includes the Sigma Factor
 recognizes promoter sequences on DNA.
' binds DNA
 binds NTPs and interacts with .
 subunits appear to be essential for assembly and
for activation of enzyme by regulatory proteins.
 ►Properties of Promoters

• Promoters typically consist of a 40 -60bp region on the 5'-side of

the transcription start site
• -40 to +20: bound by the polymerase
• -20 to +20: tightly associated with the polymerase and protected
from nuclease digestion by DNaseΙ
• Two consensus sequence elements:
– The "-35 region", with consensus TTGACA
– The Pribnow box near -10, with consensus TATAAT this
region is ideal for unwinding.

TTGACA -----16-18 bp-------TATAAT---5-8 bp---A

-35 sequence -10 sequence +1
-10 sequence (Pribonow box)

• David Prainbow
• Important in binding RNAP
• The most conserved sequence in 70 promoters at which
DNA unwinding is initiated by RNA Pol.
•The consensus sequence is TATAAT. The first two bases (TA)
and the final T are most highly conserved.
•This hexamer(6 bp sequence) is separated by between 5 and
8 bp from position +1, and the distance is critical.
-35 sequence: enhances recognition and interaction with the
polymerase s factor

• A conserved hexamer sequence around position –35

• A consensus sequence of TTGACA
• The first three positions (TTG) are the most conserved
among E. coli promoters
• Separated by 16-18 bp from the –10 box in 90% of all
promoters
the distance is critical.
Important Promoter Features

• The closer the match to the consensus the stronger the

promoter (-10 and -35 boxes)

• The absolute sequence of the spacer region (between

the -10 and -35 boxes) is not important

• The length of the spacer sequence IS important:

TTGACA - spacer (16 to 19 base pairs) - TATAAT

• Spacers that are longer or shorter than the consensus

length make weak promoters
Initiation (template recognition)

1) Binding of an RNA polymerase to the dsDNA

2) Slide to find the promoter

3) Unwind the DNA helix

4) Synthesis of the RNA strand at the start site (initiation

site), this position called position +1
There is no “zero”

Transcription Initiation Site

“Upstream” “Downstream”

-5 -4 -3 -2 -1 +1
+2 +3 +4 +5 +6
5’ 3’
3’ 5’
Direction of
transcription
Template strand
RNAP scanning a region of DNA from -40 to +20

-10 region

TTGACA…16-19 bp... TATAAT

“-35” spacer “-10”
The role of σ factor in promoter binding

• The RNA polymerase core enyzme, α2ββ’ω, has a

general non-specific affinity for DNA, which is referred to
as loose binding that is fairly stable.

• The addition of sigma factor to the core enzyme markedly

reduces the holoenzyme affinity for non-specific binding
by 20 000-fold, and enhances the holoenzyme binding to
correct promoter sites 100 times.

• Overall, sigma factor binding dramatically increases the

specificity of the holoenzyme for correct promoter-binding
site.
Elongation
Core polymerase - no sigma

• Covalently adds ribonucleotides to the 3’-end of the growing RNA

chain.
• The RNA polymerase extend the growing RNA chain in the direction
of 5’ 3’
• The enzyme itself moves in 3’ to 5’ along the antisense DNA strand.
• Polymerase is pretty accurate - only about 1 error in 10,000 bases
(not as accurate as DNAP III)
• Elongation rate is 20-50 bases per second - slower in G/C-rich
regions and faster elsewhere
• Topoisomerases precede and follow polymerase to relieve
supercoiling
Transcription bubble:

• containing ~ 17 bp of unwound DNA region and the 3’-

end of the RNA that forms a hybrid helix about 12 bp.

• moves along the DNA with RNA polymerase which

unwinds DNA at the front and rewinds it at the rear.

• The E. coli polymerase moves at an average rate of ~

40 nt per sec, depending on the local DNA sequence
Transcription bubble
RNA chain
RNA chain termination

1. Termination occurs at terminator DNA sequences.

2. The most common stop signal is an RNA hairpin (self-

complement structure) commonly GC-rich to favor the
structure stability

3. Rho-dependent and Rho-independent Termination.

Terminator

1) Rho -- independent terminator -is the termination factor protein

contains
– rho is an ATP-dependent helicase

– it moves along the RNA transcript, finds the "bubble", unwinds it

and releases the RNA chain.

A) self-complementary region that is G-C rich and can form a stem-loop or

hairpin secondary structure. GC-rich favouring the base pairing
stability and causing the polymerase to pause.
B) a run of adenylates (As) in the template strand that are transcribed into
uridylates (Us) at the end of the RNA, resulting in weak RNA-
antisense DNA strand binding.
Termination
(depends on DNA sequence - NOT a protein factor)

The A-U
base-pairing
is less stable
that favors
the
dissociation
2) Rho protein (r) dependent terminator

• Contains only the self-complementary region

• Requires r protein for termination
• r protein binds to specific sites in the single-stranded RNA
• r protein hydrolyzes ATP and moves along the nascent RNA towards
the transcription complex then enables the polymerase to terminate
transcription
• RNAP pauses at a stem-loop and r catches up to it. r unwinds DNA-
RNA hybrid causing polymerase to fall off.
• believed to break the hydrogen bonds between the template DNA and
RNA.(r has DNA-RNA helicase activity)
Cont'd
Rho-binding Site
(non-contiguous
structural features
in RNA): Stop signals
not recognized by
RNAP alone; account
for ~50% of all E. coli
terminators.

Rho: forms RNA-

dependent hexameric
helicase/ATPase,
translocates along RNA
5’-to-3’, pulling RNA
away when it reaches
the transcription
bubble.

termination
RNA polymerase/transcription and DNA polymerase/replication

RNA pol DNA pol

Template dsDNA is better than dsDNA

ssDNA

Require primer No Yes

Initiation promoter origin

elongation 40 nt/ sec 1000 bp/sec

terminator Synthesized RNA Template DNA

Transcription Regulation in Prokaryotes

• Why is it necessary?
• Bacterial environment changes rapidly.
• Survival depends on ability to adapt.
• Bacteria must express the enzymes required to survive
in that environment.
• Enzyme synthesis is costly (energetically).
• Therefore, want to make enzymes when required.