Transcription

M.PRASAD NAIDU
MSc MEDICAL BIOCHEMISTY,
Ph.D.RESEARCH SCHOLAR
Definition
Synthesis of RNA using ssDNA as a template by DNA
dependent RNA polymerase


Similar to replication in terms of chemical mechanism,
polarity, and use of template but differ in
-does not require primers
-only a short segment of DNA is transcribed

All 3 types of cellular RNAs are copied during transcription

Messenger RNAs (mRNAs) encode the amino acid sequence
of one or more polypeptides specified by a gene or set of
genes.

Transfer RNAs (tRNAs) read the information encoded in the
mRNA and transfer the appropriate amino acid to a growing
polypeptide chain during protein synthesis.

Ribosomal RNAs (rRNAs) are constituents of ribosomes, the
intricate cellular machines that synthesize proteins.

Basic Requirements of Transcription--
prokaryotic

Template ------ssDNA

Enzyme-----RNA polymerase

Regulatory proteins

Ribonucleoside triphosphates ( A, G,C,U )

Energy uses the energy released from the cleavage of
pyrophosphates to two phosphates by pyrophosphotase
Steps involved in Transcription

Initiation
-recognition of specific DNA sequence ( promoter region )
-beginning of bond formation process

Elongation


Termination & release
Initiation

Starts with the recognition of promoter sequence on the DNA
coding ( anti-template ) strand by RNA polymerase

Promoter sequence

Present on the DNA

Two common sequences are present on the upstream ( 5 ) side
of the start site

Start site is denoted by +1
Contd--
-10 sequence & -35 sequence

5------TTGACA--------TATAAT--------start site --------terminator 3
(-35 ) ( -10) +1

Sequences are 6 bp long

Distance b/n these two promoters is conserved & is a
separation of 17-19 nucleotides in normal

-10 sequence is called Pribnow box ( TATA box )

Contd

Genes with strong promoters cause frequent initiation of
transcription as often as every two seconds ( E coli ) (strong
promoters have sequence that correspond closely to the
consensus sequence )

Genes with weak promoters are transcribed about once in 10
minute ( weak promotes tend to have multiple substitution at
that site )

RNA Polymerase ( E.Coli )
Multisubunit enzyme
DNA dependent RNA polymerase
Very large molecule ( 500 kd ) and complex enzyme consisting of
four kinds of subunits
Prokaryotes have single RNA pol that transcribes all the three
RNAs ( mRNA, t RNA, r RNA )

2




------holoenzyme
RNA polymerase with out subunit is called core enzyme
Core enzyme contains the catalytic activity.



Subunit Gene Number Mass
( kd )
Role
rpo A

2 37 Binds regulatory proteins
rpo B 1 151 Forms phosphodieter bond

rpo C 1 155 Binds DNA template

rpo D 1 70 Recognizes promoter & initiates
synthesis

Contd--
RNA polymerases lack a separate proofreading 3 to 5
exonuclease active site (such as that of many DNA
polymerases)

The error rate for transcription is higher than that for
chromosomal DNA replication approximately one error for
every

10
4
to 10
5
ribonucleotides incorporated into RNA.

Contd--

After recognizing & binding to the promoter region RNA pol
synthesizes complimentary RNA sequence to the DNA template
strand ( U instead T is paired with A )

RNA synthezised from 5 to 3 ( template read from 3 to 5 )

5 end of new RNA chain is highly distinctive : a molecule starts
with either pppG or pppA ( DNA synthesis Primers )

Template strand is determined by the location of the promoter
region for that gene


Contd--

RNA pol interacts with activator & repressor proteins that
modulate the rate of transcription

In bacteria one species of RNA pol can synthezise all the RNA
molecules ( mRNA, tRNA, rRNA ) except short RNA primers
needed for DNA replication ( primase )

Elongation

RNA polymerase elongates an RNA strand by adding
ribonucleotide units to the 3-hydroxyl end

The 3-hydroxyl group acts as nucleophile, attacking the
phosphate of the incoming ribonucleoside triphosphate and
releasing pyrophosphate.

By the time 10 nucleotides are added ,the factor dissociates &
the core enzyme continues the elongation of the transcript


Contd

The template DNA strand is copied in the 3 to 5 direction
(antiparallel to the new RNA strand), just as in DNA
replication.

Each nucleotide in the newly formed RNA is selected by
Watson-Crick base-pairing interaction

RNA pol
(NMP)n + NTP (NMP)n+1 + PPi
RNA Lengthened RNA

Termination
E. coli has at least two classes of termination signals:
one class relies on a protein factor called (rho)
the other is independent.


Most independent terminators have two distinguishing
features.
The first is a region that produces an RNA transcript with self
complementary sequences, permitting the formation of a
hairpin structure centered 15 to 20 nucleotides before the
projected end of the RNA strand.
Contd--
The second feature is a highly conserved string of A residues
in the template strand that are transcribed into U residues
near the 3 end of the hairpin.

When a polymerase arrives at a termination site with this
structure, it pauses

Formation of the hairpin structure in the RNA disrupts
several AUU base pairs in the RNA-DNA hybrid segment and
may disrupt important interactions




Contd

Rho ( ) dependent termination

Rho factor is an ATP dependent RNA-DNA helicases

Recognizes and bind to the termination signals and disrupts
the nascent RNA/DNA complex
Transcription Is Regulated at Several Levels

Requirements for any gene product vary with cellular
conditions or developmental stage, and transcription of each
gene is carefully regulated to form gene products only in the
proportions needed.

Regulation can occur at any step in transcription, including
initiation ,elongation and termination.

Much of the regulation is directed at the polymerase binding
and transcription initiation

The binding of proteins to sequences both near to and distant
from the promoter can also affect levels of gene expression.

Protein binding can activate transcription by facilitating either
RNA polymerase binding or steps further along in the
initiation process, or it can repress transcription by blocking
the activity of the polymerase.

In E. coli, one protein that activates transcription is the cAMP
receptor protein (CRP), which increases the transcription of
genes coding for enzymes that metabolize sugars other than
glucose when cells are grown in the absence of glucose.

Contd--

Repressors are proteins that block the synthesis of RNA at
specific genes.

In the case of the Lac repressor , transcription of the genes
for the enzymes of lactose metabolism is blocked when
lactose is unavailable
Inhibitors
Rifampicin ( antitubercular
drug)
-semisynthetic derivative
( sterptomyces)
-specifically inhibit initiation not by
blocking the binding of RNA pol
but rather by interfering with
the formation of the first
phosphodiester bond
-Site of action is on subunit of
RNA pol
-some mutants having an altered
subunit are resistant to
rifampacin
Actinomycin D
-polypeptide derived from streptomyces

-binds tightly & specifically to double stranded DNA & prevents
it from being an effective template for RNA synthesis

-at low concentration inhibits transcription without appreciably
affecting replication & protein synthesis

-inhibitor of both prokaryotic & eukaryotic cell

-effective therapeutic agent in the treatment of some cancers