According to the central dogma of molecular biology, genetic information usually flows (1) from DNA to DNA during its transmission from generation to generation and (2) from DNA to protein during its phenotypic expression in an organism

1. Dr. Pawan Kumar Kanaujia
Assistant Professor
Molecular Biology
(Theory)
Prokaryotic Transcription
& gene structure

2. Transfer of Genetic Information: The Central Dogma
According to the central dogma of molecular biology,
genetic information usually flows (1) from DNA to DNA
during its transmission from generation to generation and
(2) from DNA to protein during its phenotypic expression
in an organism
The transfer of genetic information from DNA to protein
involves two steps:
(1) transcription, the transfer of the genetic information
from DNA to RNA, and
(2) translation, the transfer of information from RNA to
protein.

3. Fig: The flow of genetic information according to the central dogma of molecular
biology. Replication, transcription, and translation occur in all organisms; reverse
transcription occurs in cells infected with certain RNA viruses. Not shown is the
transfer of information from RNA to RNA during the replication of RNA viruses.

4. GENERAL FEATURES OF RNA SYNTHESIS
RNA synthesis occurs by a mechanism that is similar to
that of DNA synthesis except that:
(1) The precursors are ribonucleoside triphosphates
rather than deoxyribonucleoside triphosphates.
(2) Only one strand of DNA is used as a template for the
synthesis of
a complementary RNA chain.
(3) RNA chains can be initiated de novo, without any
requirement for a
pre-existing primer strand.
The RNA molecule produced will be complementary and
antiparallel to the DNA template strand and identical,
except that uridine residues replace thymidines, to the
DNA nontemplate strand.

5. Fig: The RNA chain elongation reaction catalyzed by RNA polymerase

6. A single RNA polymerase carries out all transcription in
most prokaryotes, whereas five different RNA polymerases
are present in eukaryotes, with each
polymerase responsible for the synthesis of a distinct class
of RNAs.
RNA synthesis takes place within a locally unwound
segment of DNA, sometimes called a transcription bubble,
which is produced by RNA polymerase. The nucleotide
sequence of an RNA molecule is complementary to that of
its DNA template strand except that uracil replaces thymine.
RNA polymerases bind to specific nucleotide sequences
called promoters, and with the help of proteins called
transcription factors, initiate the synthesis of RNA
molecules at transcription start sites near the promoters.

7. Transcription in Prokaryotes
A segment of DNA that is transcribed to produce one RNA
molecule is called a
transcription unit. Transcription units may be equivalent
to individual genes,
or they may include several contiguous genes. Large
transcripts that carry the coding sequences of several
genes are common in bacteria. The process of
transcription can be divided into three stages:
(1) Initiation of a new RNA chain,
(2) Elongation of the chain, and
(3) Termination of transcription and release of the
nascent RNA molecule.

8. Fig: The three stages of transcription: initiation, elongation, and termination

9. RNA POLYMERASES: COMPLEX ENZYMES
Bacterial RNA polymerase: Bacterial cells typically possess only one type of RNA
polymerase, which catalyzes the synthesis of all classes of bacterial RNA: mRNA,
tRNA, and rRNA.
Fig: In bacterial RNA polymerase, the core enzyme consists of five subunits: two
copies of alpha (α), a single copy of beta (β), a single copy of beta prime (β′), and a
single copy of omega (ω). The core enzyme catalyzes the elongation of the RNA
molecule by the addition of RNA nucleotides. (a) The sigma factor (σ) joins the core
to form the holoenzyme, which is capable of binding to a promoter and initiating
transcription. (b) The molecular model shows RNA polymerase (in yellow) binding
DNA.
(b)

10. RNA Polymerase Holoenzyme
Subunit (s) Function (s)
alpha (α)
(two copies)
The subunits are involved in the assembly of the of core
RNA polymerase
beta (β) subunit contains the ribonucleoside triphosphate binding
site
beta prime (β′) subunit harbors the DNA template-binding region
omega (ω) The ω subunit is not essential for transcription, but it helps
stabilize the enzyme
sigma factor (σ) The sigma (σ) factor recognize the promoter and controls
the binding of RNA polymerase to the promoter. Sigma is
required only for promoter binding and initiation; when a
few RNA nucleotides have been joined together, sigma
usually detaches from the core enzyme

11. Fig: In bacterial promoters, consensus sequences are found upstream of the
start site, approximately at positions −10 and −35.

12. The most commonly encountered consensus sequence, found in
almost all bacterial promoters, is centered about 10 bp upsteam
of the start site. Called the –10 consensus sequence or,
sometimes, the Pribnow box, its consensus sequence is
5′–T A T A A T–3′
3′–A T A T T A–5′
Another consensus sequence common to most bacterial
promoters is
5′–T T G A C A–3′
3′–A A C T G T–5′
, which lies approximately 35 nucleotides upstream of the start
site and is termed the −35 consensus sequence

13. In the case of Escherichia coli, the predominant σ factor is
called σ 70
Promoters recognized by RNA polymerase containing σ 70
share the following characteristic structure:
Two conserved sequences, each of 6 nucleotides,
separated by a nonspecific stretch of 17–19 nucleotides.
The two defined sequences are centered, respectively, at -
10 bp and at -35 bp upstream of the site where RNA
synthesis starts.
The sequences are thus called the –35 (minus 35) and –
10 (minus 10) regions, or elements, according to the
numbering scheme.
DNA nucleotide encoding the beginning of the RNA chain
is designated +1.

14. Features of bacterial promoters. Various combinations of
bacterial promoter elements are shown

15. Fig b. An additional DNA element that binds RNA polymerase is found in
Some strong promoters, for example, those directing expression of the
ribosomal RNA (rRNA) genes. This is called an UP-element and increases
polymerase binding by providing an additional specific interaction between
the enzyme and the DNA.
Fig c. Another class of σ 70 promoters lacks a –35 region and instead has a
so-called “extended –10” element. This comprises a standard –10 region
with an additional short sequence element at its upstream end. Extra
contacts made between polymerase and this additional sequence
element compensate for the absence of a –35 region.
Fig d. A final DNA element that binds RNA polymerase is sometimes found
just downstream from the –10 element. This element is called the
discriminator. The strength of the interaction between the discriminator
and polymerase influences the stability of the complex between the
Enzyme and the promoter.

16. Fig: σ and α subunits recruit RNA polymerase core enzyme to the promoter.
The carboxy-terminal domain of the a subunit (αCTD) recognizes the UP-
element (where present), whereas σ regions 2 and 4 recognize the –10 and –35
regions, respectively. In this figure, RNA polymerase is shown in a schematic
representation rather different from that presented in earlier figures.
An additional DNA element that binds RNA polymerase is found in some
strong promoters, for example, those directing expression of the ribosomal
RNA (rRNA) genes. This is called an UP-element and increases polymerase
binding by providing an additional specific interaction between the enzyme and
the DNA.

17. Structure of the holoenzyme

18. Fig: Channels into and out of the open complex. This figure shows the
relative positions of the DNA strands (template strand in gray, non
template strand in orange), the four regions of σ, the –10 and –35 regions
of the promoter, and the start site of transcription (+1). The channels
through which DNA and RNA enter or leave the RNA polymerase enzyme
are also shown. The only channel not shown here is the nucleotide entry
(NTP-uptake) channel, through which nucleotides enter the active site
cleft for incorporation into the RNA chain as it is made. As drawn, that
channel would enter the active site from the back of the page at about
the position shown as “+1” on the DNA. Where a DNA strand passes
underneath a protein, it is drawn as a dotted ribbon. σ region 3/4 linker—
also called σ 3.2—is the linker region between σ 3.1 and σ 4.

19. A channel runs between the pincers of the claw-shaped enzyme
The active site of the enzyme, which is made up of regions from both
the β and β′ subunits, is found at the base of the pincers within the
active center cleft.
There are five channels into the enzyme, as shown in the illustration of
the open complex:
1). The NTP-uptake channel allows ribonucleotides to enter the active
center (not shown)
2). The RNA-exit channel allows the growing RNA chain to leave the
enzyme as it is synthesized during elongation.
3). The downstream DNA (i.e., DNA ahead of the enzyme, yet to be
transcribed) enters the active center cleft in double-stranded form
through the downstream DNA channel (between the pincers).
4). The non-template strand exits the active center cleft through the
non-template-strand (NT) channel and travels across the surface of the
enzyme.
5). The template strand, in contrast, follows a path through the active
center cleft and exits through the template-strand (T) channel.

20. During Initial Transcription, RNA Polymerase Remains Stationary
and Pulls Downstream DNA into Itself
RNA polymerase produces and releases short RNA transcripts of ,
10 nucleotides (abortive synthesis) before escaping the promoter,
entering the elongation phase, and synthesizing the proper transcript.
Three general models were proposed
1). The “transient excursion” model proposes transient cycles of forward and
reverse translocation of RNA polymerase. Thus, polymerase is thought to leave
the promoter and translocate a short way along the DNA template, synthesizing
a short transcript before aborting transcription, releasing the transcript, and
returning to its original location on the promoter.
2). “Inchworming” invokes a flexible element within the polymerase that allows
a module at the front of the enzyme, containing the active site, to move
downstream, synthesizing a short transcript before aborting and retracting to
the body of the enzyme still at the promoter.
3).“Scrunching” proposes that DNA downstream from the stationary, promoter-
bound, polymerase is unwound and pulled into the enzyme. The DNA thus
accumulated within the enzyme is accommodated as single-stranded bulges.

21. Mechanism of initial transcription

22. Fig: During initial transcription, the active center of RNA polymerase is
translocated forward relative to the DNA template and synthesizes short
transcripts before aborting, then repeats this cycle until it escapes the
promoter.
Three models have been proposed to account for this and are
shown in the figure.
1. Transient excursions- polymerase moves along the DNA.
2. Inchworming- the front part of the enzyme moves along the DNA,
but because of a flexible region within the enzyme, the back part of the
enzyme can remain stationary at the promoter.
3. Scrunching –the enzyme remains stationary and pulls the DNA into
itself. As is the evidence supporting scrunching as the true picture of
what goes on during transcription.

23. Promoter Escape Involves Breaking Polymerase–Promoter Interactions
and Polymerase Core–σ Interactions
During initial transcription, the process of abortive initiation takes place,
and short—9 nucleotides or shorter—transcripts are generated and released.
Polymerase manages to escape from the promoter and enter the
elongation phase only once it has managed to synthesize a transcript of a
threshold length of 10 or more nucleotides.
Once this length, the transcript cannot be accommodated within the region
where it hybridizes to the DNA and must start threading into the
RNA exit channel.

24. The Elongating Polymerase Is a Processive Machine That Synthesizes
and Proofreads RNA
DNA passes through the elongating enzyme in a manner very similar to its
passage through the open complex. Thus, double-stranded DNA enters the
front of the enzyme between the pincers. At the opening of the catalytic
cleft, the strands separate to follow different paths through the enzyme
before exiting via their respective channels and re-forming a double helix
behind the elongating polymerase.
Ribonucleotides enter the active site through their defined channel and
are added to the growing RNA chain under the guidance of the template
DNA strand.
Only 8 or 9 nucleotides of the growing RNA chain remain base-paired to
The DNA template at any given time; the remainder of the RNA chain is
peeled off and directed out of the enzyme through the RNA exit channel.

25. 1. pyrophosphorolytic editing. In this, the enzyme uses its active site,
A simple back-reaction to catalyze the removal of an incorrectly
inserted ribonucleotide
 Reincoporation of PPi
The enzyme then incorporate another ribonucleotide in the growing
RNA chain
Remove incorrect bases
As well as synthesizing the transcript, RNA polymerase performs two
proofreading functions on that growing transcript.
Proofreading functions
Hydrolytic editing is stimulated by Gre factor:
which both enhance hydrolytic editing function and serve as
elongation stimulating factors
that is, they ensure that polymerase elongates efficiently and help
overcome “arrest” at sequences that are difficult to transcribe.
2. hydrolytic editing, the polymerase backtracks by one or more nucleotides
and cleaves the RNA product, removing the error-containing sequence.

26. Transcription Is Terminated by Signals within the RNA Sequence
In bacteria, terminators are two types:
1st -Rho-dependent requires a protein called Rho to induce termination.
2nd -Rho-independent terminators, also called intrinsic terminators
Because they need no other factors to work, consist of two sequence
elements: a short inverted repeat (of ~20 nucleotides) followed by a stretch
of about eight A:T base pairs.
These elements do not affect the polymerase until they have been
transcribed—that is, they function in the RNA rather than in the DNA.
When polymerase transcribes an inverted repeat sequence, the resulting
RNA can form a stem-loop structure (often called a “hairpin”) by
base-pairing with itself.
Formation of the hairpin causes termination by disrupting the elongation
complex. That is, the hairpin induces termination by either pushing
polymerase forward, or inducing a conformational change in
polymerase.

27. Sequence of a Rho-independent terminator
Fig: At the top is the sequence, in the DNA, of the terminator.
Below is shown the sequence of the RNA, and the bottom image shows
the structure of the terminator hairpin.

28. Transcription termination

29. Fig: A model for how the Rho-independent terminator might work.
(Top) The hairpin forms in the RNA as soon as that region has been
transcribed by polymerase (the enzyme is not shown here).
(Middle) That RNA structure disrupts polymerase just as the enzyme is
transcribing the AT-rich stretch of DNA downstream.
(Bottom) Hairpin disrupts the transcribing polymerase and the weak
interactions between the transcript and the template DNA appear to
make release of that transcript easier.

30. Fig: The three stages of transcription: initiation, elongation, and termination