TRANSCRIPTION IN PROKARYOTES (BACTERIA)

Transcription or RNA synthesis is the process of transcribing DNA nucleotide sequence

information into RNA sequence information. Transcription is catalyzed by a large enzyme called
RNA polymerase.

RNA polymerase: RNA polymerase from E.coli is a very large (~ 400kd) and complex enzyme
constituting four kinds of subunits.

Subunit Gene No. of Mol.wt

genes
α rpoA 2 37kd
β rpoB 1 151kd
β’ rpoC 1 155kd
σ rpoD 1 70kd

The subunit composition of the entire enzyme called holoenzyme is made up of αββ’σ. The σ
subunit helps to find a promoter site to start transcription. It also participates in the initiation of RNA
synthesis. RNA polymerase without this σ sub unit is called core enzyme. The core enzyme
contains the catalytic site.

RNA polymerase performs the following multiple functions:

It searches DNA for initiation sites called promoter sequence.
It unwinds a short stretch of double helical DNA to produce a single stranded DNA template
from which it takes information.
It selects the correct ribo nucleotide triphosphate and catalyses the formation of phosphor
diester bond. This process is repeated many times as the enzyme moves unidirectional along
the DNA templates, where the RNA chain grows in the 5’ → 3’ direction.
RNA polymerases in contrast to DNA polymerase cal initiate chain growth without primers.
The synthesis of RNA consists of four discrete stages:
Binding of RNA polymerase to DNA template at a specific site (promoter),
Initiation,
Chain elongation,
Chain termination and release.
Binding of RNA polymerase to DNA template at a specific site:

The first step in transcription is the binding of RNA polymerase to a DNA molecule. Binding
occurs in particular regions called promoters, which are sequences of DNA that dictates RNA
polymerases to bind at proper initiation site. The first DNA nucleotide to be transcribed is denoted
as +1 and the nucleotide preceding the start site as -1. The σ subunit enables the RNA polymerase
to recognize the promoter sites. The promoter site is encountered by an RNA polymerase by a
random walk on the DNA. In case of E.coli the predominant σ factor is called σ70. Promoters
recognized by RNA polymerase containing σ70 shares the following characteristic structure.

The promoter sequences contains 6 bases (TTGAGA), whish is not transcribed. It is found
approximately 35 base pairs before the transcription start site. TATA box lies within the promoter
sequence about 10 base pairs before the start site.

Two conserved sequences each of six nucleotides are separated by a nonspecific stretch of
17 to 19 nucleotides. These sequences are present upstream of the start site at -10 and -35
regions.

TATAATG START SITE

5’ -35 -10 3’

(Pribnow box) (TATA box)

After binding of the holoenzyme to the pribnow box the conformation called open
promoter complex is formed by local unwinding starting about 10bp. From the left of the pribnow
box and extending about 20bp past the position of the first transcribed base, RNA polymerase itself
induces the unwinding. This melting of DNA is necessary for pairing of incoming ribonucleotides.

σ sub unit Unwound DNA(transcription bubble)

(12 bps opened)

RNA polymerase promoter sequence

INITIATION:
RNA synthesis can start from 5’ to 3’ direction with new nucleotides being added to the 3’ end
of the growing chain. The RNA polymerase opens or unwinds the double helix to form a
transcription bubble, about 12 to 20 base pairs in length. Once the open promoter complex is
formed; RNA polymerase is ready to initiate synthesis.

The RNA polymerase recognizes pribnow box and TATA sequences, binds to the promoter,
and unwinds a short segment of DNA beginning around the pribnow box. Transcription starts 6 or 7
base away from the 3’ end of the promoter. The RNA polymerase remains at the promoter while it
constructs a chain about 9 nucleotides long, then it begin to move down the template strand.

Once the RNA synthesis begins, the sigma factor dissociates from the core enzyme – DNA
complex and is available to aid another core enzyme. There are several different sigma (σ) factors
in E.coli; σ70 is most often involved in transcription initiation. α subunit seems to be involved in the
assembly of the core enzyme, region of promoters.

RNA polymerase contains two nucleotide binding sites. One is the initiation site and the other
is elongation site. The initiation site primarily binds with purine triphosphates (ATP and GTP). ATP
is usually the first nucleotide in the growing chain. Thus the first DNA base that is transcribed is
thymine. The initiating nucleotide triphosphate binds to the enzyme in the open promoter complex
and forms a hydrogen bond with the complementary DNA base.

The elongation site is then filled with a nucleotide triphosphate that is selected by its ability to
hydrogen bond with the next base in the DNA strand. The two nucleotides are joined together by a
phosphodiester bond, the first base is released from the initiation site and the initiation is complete.
The dinucleotide remains hydrogen bonded to the DNA. The elongation phase then begins when
the polymerase releases the base and then moves along the DNA chain.

CHAIN ELONGATION:

After several nucleotides are added (Eight is the best estimate) to the growing chain, RNA
polymerase undergoes a conformational change and loses the σ sub unit. Then elongation is
carried out by core enzyme. The core enzyme moves along the DNA, a nucleotide triphosphate
pairs with the next DNA base and opening the DNA helix as it moves.
 The DNA helix closes as the synthesis proceeds. The newly synthesized RNA is
released from its hydrogen bonds with the DNA as the helix reforms. Roughly 12 RNA bases are
paired to the DNA in the open region. The rate of elongation is about 50 nucleotide addition per
second.

Template strand RNA polymerase Coding strand Unwinding

Rewinding

Nascent RNA 5’ ppp RNA - DNA hybrid helix

CHAIN TERMINATION AND RELEASE:

Termination of transcription is precisely controlled as initiation. In termination, the formation
of phosphodiester bond ceases, RNA - DNA hybrid dissociates the melted region of DNA rewinds
and RNA polymerase releases the DNA.
During termination, RNA polymerase recognizes the TER sequences of template DNA that
codes for RNA stretch that can hydrogen bond to form a hairpin - shaped loop and stem structure.
This structure appears to stop transcribing DNA.
There are two ways by which termination occurs:
Rho independent termination
Rho dependent termination

Rho independent termination

The transcribed region of DNA templates contains stop signals. The simplest one is a
palindromic AC rich region followed by AT rich region. The RNA transcript of this DNA palindrome is
self complementary. Hence its bases can form a hairpin structure with a stem and a loop having
high GC content. This stable hairpin is followed by a sequence of four or more uracil residues. The
RNA transcript ends within or just after them.
 RNA polymerase pauses immediately after it has synthesized a stretch of RNA that folds into
a hairpin. The RNA - DNA hybrid helix produced after the hairpin is unstable. Hence the pause in
the transcription permits the weakly bound nascent RNA to dissociate from the DNA template.
Rho dependent termination

DNA template

mRNA tail
ATP + H20
Rho protein
ADP + Pi

Rho protein is an ATP dependent helicase that binds to the nascent RNA chain and pulls it
away from RNA polymerase and the DNA template. Rho factor attaches with recognition site of
mRNA and moves along RNA following RNA polymerase. Now RNA polymerase pauses at
terminator and Rho catches up. Rho factor unwinds DNA - RNA hybrid in transcription bubble.
 TRANSCRIPTION IN EUKARYOTES

Transcription in eukaryotes is undertaken by polymerases closely related to RNA

polymerases found in bacteria. Eukaryotes have three different RNA polymerases.
Polymerase I
Polymerase II
Polymerase III (whereas prokaryotes only one)
Several initiation factors are required for efficient and promoter - specific initiation in
eukaryotes called General Transcription Factors (GTFs).

RNA polymerases - II core promoters:

The eukaryotic core promoter refers to the minimal set of sequence elements for accurate
transcription by the polymerase II machinery.

A core promoter is usually 40 nucleotides long, Extending either upstream or downstream of

the transcription start site. The promoter sequence consists of following elements:
TF IIB recognition element (BRE)
TATA box
The initiator (INR)
Down stream promoter element (DPE)

Beyond these, there are other regulatory sequences which are required for efficient
transcription.

TFIIB TBP TFIID TFIID

-37 -32/-31 -26 -2 +4 +28 +32

BRE bTvBRB

BRE TATA INR DPE

A typical structure of eukaryotic promoter sequence

 The regulatory sequences can be grouped in various categories depending on their location,
organism and function. These include:
 Promoter proximal elements
 Upstream activator sequences (UPAS)
 Enhancers
 Series of repressing elements
1. Silencers
2. Boundary elements
3. Insulators
All these DNA elements binds with regulatory proteins (activators and repressors), which
helps or hinder transcription from the core promoters.

The actual process of transcription mainly involves in three steps:

1. Initiation
2. Elongation
3. Termination

INITIATION:

FORMATION OF PRE INITIATION COMPLEX WITH GENERAL TRANSCRIPTION FACTORS

(GTFS) AT THE PROMOTER SEQUENCES

The GTFs collectively performs the functions that are done by σ factors in bacterial
transcription. Thus, they help polymerase to bind in promoter sequence and melt the DNA. They
also help polymerase to escape from the promoter and shifted to elongation phase.

The complete set of general transcription factors (GTFs) and polymerase bound together at
the promoter and poised for initiation called Pre Initiation Complex. This complex formation begins
at the TATA box, which is recognized by the GTF - TF II D complex. The component of TF II D that
binds to TATA box is TBP (TATA Binding Protein). Upon binding DNA, TBP extensively distorts the
TATA sequence. The TBP - DNA complex provides a platform to recruit other general transcription
factors and polymerase itself to the promoter.
 The step wise assembly of Pol II pre - initiation complex is shown above. Once assembled at
the promoter, pol II leaves the pre - initiation complex upon addition of the nucleotide precursors
required for RNA synthesis and after phosphorylation of serine residues with in the enzymes ‘tail’.
The tail contains multiple repeats of hepta peptide sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

There are 27 of these repeats in yeast Pol II and 52 in Human. Each repeat contains sites for
phosphorylation by specific kinases including one that is a subunit of TF II H.

ELONGATION:

Once polymerase has initiated transcription phase, it shifts in to the elongation phase. This
involves the Pol II enzyme shedding most of its initiation factors. In this phase another set of factors
is recruited. These are called elongation factors. Thus phosphorylation of CTD leads to the
exchange of initiation factors for those required for elongation and processing.

Various proteins also stimulate elongation by Pol II.

Kinases - P (TEFB) phosphorylates the serine residues at 2 nd
position of CTD. It also activates Heat Shock Protein (HSPT -
5) and transcription activator (TAT - SF1) protein.
TF II S - stimulates over all rate of elongation and contributes
proof reading by polymerase.

Once transcribed, eukaryotic RNA has to be processed in various ways before being exported
from the nucleus where it can translated. They are,
 Capping as 5’ end of RNA
 Splicing (Removal of introns)
 Polyadenylation at 3’ end of RNA

The first RNA processing event is capping. This involves the addition of modified guanine base
to the 5’ end of the RNA. Specifically it is methylated guanine and is joined to the RNA transcript by
an unusual 5’- 5’ linkage involving three phosphates. The enzymes involved in capping are, i) RNA
triphosphatase ii) Guanyl transferase iii) Methyl transferase. The second processing event is RNA
splicing is done by small nuclear RNAs called snRNAs.
TERMINATION:

Polyadenylation of the 3’ end of the mRNA is ultimately linked with the termination of the
transcription. Once the polymerase has reached end of a gene, it encounters specific sequences
that, after being transcribed in to RNA, triggers the transfer of Polyadenylation enzymes to that
RNA, leading to,

1. Cleavage of messager RNA,

2. Addition of many adenine residues to 3’ end,
3. Termination of transcription by polymerase.

Once CPSF & CSTF bound to RNA, other proteins are recruited as well, leading initially to RNA
cleavage and then Polyadenylation occurs.