Mechanisms of Transcription

GENETIC MATERIAL
Organization Protection Replication Expression
DNA bases
RNA
Protein
Transcription
Translation
Objectives
Understand the structure of RNA polymerases

Understand the phases of the transcription cycle

Understand the differences between transcription and replication

Understand differences between prokaryotic and eukaryotic
transcription

Transcription is, chemically and enzymatically,
very similar to DNA replication, but there are
some important differences:
RNA is made of ribonucleotides (rather than deoxyribonuleotides)

RNA polymerase catalyzes the reaction (does not need a primer)

The synthesized RNA does not remain base-paired to the template DNA
strand

Less accurate (one in 10000,compared to one in 10000000 for replication)

Transcription selectively copies only certain parts of the genome and makes
one to several hundred, or even thousand, copies of any given section of the
genome. (replication must copy the entire genome and do so once every cell
division)
Transcription of the DNA into RNA.(in the absence of the enzymes involved)
Multiple RNA polymerases can transcribe the same gene at the same time.
A cell can synthesize a large number of RNA transcription in a short time.
Topic 1:

RNA Polymerase and The
Transcription Cycle
RNA Polymerases Comes in Different Forms, but
Share Many Features.
1.RNA polymerases performs essentially the same reaction in the cell,from
bacteria to humans.

2.The cellular RNA polymerases are made up of multiple subunits.

3.Bacteria have only a single RNA polymerases ,while in eukaryotic cells there
are three: RNA Pol .

4.Pol is the polymerases responsible for transcribing most genesindeed,
essentially all protein-encoding genes.

5.Pol and Pol are each involved in transcribing specialized, RNA-encoding
genes.

Comparison of the crystal structures of prokaryotic
and eukaryotic RNA polymerases.
The shape of each enzyme resembles a crab claw.
Transcription by RNA polymerases Proceeds in a Series
of steps
Initiation.

Elongation.

Termination.
Initiation
A promoter is the DNA sequence that initially binds the RNA polymerase.
Promoter-polymerase complex undergoes structural changes required for
initiation to proceed .
The base at the transcription site unwinds and producing a bubble of
single-stranded DNA.
Transcription always occurs in a 5 to 3 direction.
Only one strand of DNA acts as a template.
For a given promoter the same strand is always transcribed
Elongation
Once RNA polymerase synthesizes approximately 10 bases of RNA it shifts
into elongation phase
This transition requires further conformational change- tight gripping of the
template
During elongation RNA polymerase performs following functions:
A. RNA catalysis
B. Unwinds DNA in front and reanneals it behind
C. Dissociates growing RNA chain
D. Performs proofreading
Termination
Once the polymerase has transcribed the length of the gene (or genes), it must
stop and released the RNA product. This step is called termination.

In some cells, termination occurs at the specific and well-defined DNA
sequences called terminators. Some cells lack such termination sequences.
Transcription Initiation Involves three Defined
Steps
The initial binding of polymerase to promoter to form what is called a closed
complex: DNA double stranded and enzyme bound to it
The closed complex undergoes a transition to the open complex: DNA strand
separates over a distance of around 14 bp around the start site to form a
transcription bubble
First two nucleotides brought into the active site, aligned to the template and
then joined together. This followed by enzyme movement on the template
strand
Incorporation of around first 10 nucleotides is inefficient and often at this stage
enzyme releases short transcripts.
Once a enzyme gets further than the 10 bp it starts synthesizing RNA
efficiently. This process is also known as promoter escape

Topic 2:
The Transcription Cycle in
Bacteria
Bacteria Promoters Vary in Strength and Sequence,
but Have Certain Defining Features
The bacteria core RNA polymerases can, in principle, initiate transcription
at any point on a DNA molecule.
In cells, polymerases initiate transcription only at promoters.
An initiation factor called that converts core enzyme into the form that
initiates only at promoter. That form of the enzymes is called the RNA
polymerase holoenzyme.
In the case of E.coli, the predominant is called 70. Promoter recognized by
70 contains two conserved sequences (-35 and 10 regions/elements)
separated by a non-specific stretch of 17-19 nt.
Position +1 is the transcription start site
Features of bacteria promoters. Various combination of bacteria promoter elements are shown.

a. 70 promoters contain recognizable 35 and 10 regions, but the sequences are not identical.

b. UP-element is an additional DNA elements that increases polymerase binding by providing the
additional interaction site for RNA polymerase.

c. Another class of s70 promoter lacks a 35 region and has an extended 10 element
compensating for the absence of 35 region.
The Factor Mediate Binding of Polymerase to the
Promoter
Regions of Those regions factor that
recognize specific regions of the promoter
are indicated by arrows. Region 2.3 is
responsible for melting the DNA.
2 binds to -10 and 4 binds to -35

4- helix turn helix, 2- helix

-35 in mere binding

-10 binding and transcription initiation,
DNA melting initiated in this region

3 binds to extended -10
and subunits recruit RNA polymerases core Enzyme to the promoter. The C-domain of the
subunit ( CTD) Recognize the UP-element, while region 2 and 4 recognize the -10 and -35
regions respectively.
UP-element not recognized by , instead by carboxyl terminal domain of
subunit of RNA polymerase
Transition to the Open Complex Involves Structural
Changes in RNA Polymerase and in the Promoter DNA
Transition from closed to open complex (also known as isomerization)
requires DNA melting between positions -11 and +3. Isomerization is
independent of ATP hydrolysis

After Isomerization transcription is essentially irreversible; Formation of closed
complex is reversible

Two striking structural changes during Isomerization:
A. Pincers at the front end of the enzyme clamp down tightly on the downstream DNA
B. factor N-terminal region (region 1.1) lies in the path of DNA in closed complex.
During isomerization this region shifts some 50 A and gets away from the path of
incoming DNA. This ensures that DNA is accessible to the active centre.
Transcription is Initiated by RNA Polymerase without
the Need for a Primer
DNA polymerase can only extend an existing polynucleotide chain.

However, RNA polymerase can initiate a new RNA chain on a DNA template
and does not need a primer.
RNA Polymerase Synthesizes Several Short RNAs
Before Entering the Elongation Phase
Abortive initiation: enzyme synthesizes short RNA molecules of less than 10
nucleotides in length.

factor region 3.2 lies in the middle of RNA exit channel in the open complex
and to make RNAs longer than 10 nucleotides, this region should be ejected
from that location. This process of ejecting sometimes require many attempts.

After ejection of region 3.2, factor becomes weakly associated with the
elongating enzyme and it is often lost from the elongating complex.

The Elongating Polymerase is a Processive Machine that
Synthesizes and Proofreads RNA
DNA separated at catalytic cleft, NT and T strands follow different paths, before
exiting via their respective channels to reanneal. In this process RNA gets
synthesized using nucleotides and template strand as guide.

Only 8-9 nucleotides of growing RNA chain remain base paired; remainder of
the RNA chain is peeled off and exits through RNA exit channel.

Two proofreading functions:
Pyrophosphorolytic editing: simple back reaction to catalyze removal of an incorrectly
inserted ribonucleotide, by incorporation of PPi. This is then replaced by a correct
nucleotide.
Hydrolytic editing: Polymerase backtracks by one or more nucleotides and cleaves
the RNA product, thereby removing the error-containing sequence.
Transcription is terminated by signals within the RNA
sequence
Terminators: the sequence that trigger the elongating polymerases to
dissociate the DNA and release the RNA chain it has made.

In bacteria, terminators come in two type: Rho-independent (also called as
intrinsic terminator) and Rho-dependent.

Rho-independent causes polymerase to terminate without involvement of other
factors, whereas Rho-dependent termination need Rho protein.
Transcription termination. Shown is a model for how
the Rho-independent terminator might work.