RNA Polymerase Enzyme

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Introduction
The Central Dogma of Molecular Biology

DNA

RNA

Protein
1. The DNA replicates its information in
a process that involves many enzymes:
replication.
2. The DNA codes for the production of
messenger RNA (mRNA) during
transcription.
3. In eucaryotic cells, the mRNA is
processed (essentially by splicing) and
migrates from the nucleus to the
cytoplasm.
4. Messenger RNA carries coded
information to ribosomes. The
ribosomes "read" this information and
use it for protein synthesis. This
process is called translation.
• Proteins do not code for the
production of protein, RNA or DNA.
They are involved in almost all
biological activities, structural or
enzymatic.
Transcription
Transcription is the process by which a single stranded RNA is
formed from a single strand of DNA.
The process involves :
1. Uncoiling of the 2 strands of DNA in a specific region. It
exposes the bases of the DNA strands.
2. One strand of DNA remains dormant & the other one acts
as the template for the formation of the new RNA strand.
3. The building blocks the free nucleotides align themselves
& form the complementary RNA according to the base
pairing rule.
4. The reaction is catalyzed by the enzyme RNA
Polymerase.
5. RNA polymerase catalyzes the formation of
phosphodiesterase bonds between nucleotides (using tri-
phosphate nucleotides).
6. RNA polymerase moves stepwise along the DNA
extending the RNA chain as it goes.
7. As RNA polymerase moves it unwinds the next part of the
helix, the helix behind closes & the mRNA is displaced
mRNA Strands

mRNA is always synthesized 5’ to 3’ on a 3’ to 5’ template

5’ 3’
Coding strand
(nontemplate)

3’ 5’

Sense strand
(template)

5’ 3’
• RNA polymerase (RNAP or
RNApol) is an enzyme that
makes a RNA copy of a DNA or
RNA template. In cells, RNAP
is needed for constructing
RNA chains from DNA genes, a
process called transcription.

• RNA polymerase enzymes are

essential to life and are found
in all organisms and many
viruses. In chemical terms,
RNAP is a nucleotidyl
transferase that polymerizes
ribonucleotide at the 3’ end of
an RNA transcript.
History
• RNA Polymerase or
RNAP was discovered
independently by Sam
Weiss & Jerard Hurwitz in
1960.
• In 2006, the Nobel Prize
in Chemistry was
awarded to Roger
Kornberg for creating
detailed molecular images
of RNA polymerase
during various stages of
transcription.
Structure Of RNA
Polymerase
RNA polymerase in Prokaryotes (Bacteria) :
In bacteria, the same enzyme catalyzes the
synthesis of mRNA
• RNAP is a relatively large molecule.
The core enzyme has 5 subunits (~400
kDa):α2: the two α subunits assemble
the enzyme and recognize regulatory
factors. Each subunit has two
domains:
o αCTD (C-Terminal domain) binds
the UP element of the extended
promoter, and αNTD (N-terminal
domain) binds the rest of the
polymerase.
o β this has the polymerase activity
(catalyzes the synthesis of RNA)
which includes chain initiation and
elongation.
o β': binds to DNA (nonspecifically).
o ω: restores denatured RNA
polymerase to its functional form in
vitro.
• In order to bind promoter-
specific regions, the core
enzyme requires another
subunit, sigma (σ). The sigma
factor greatly reduces the
affinity of RNAP for nonspecific
DNA while increasing
specificity for certain promoter
regions, depending on the
sigma factor.Thus, transcription
is initiated at the right region.
The complete holoenzyme
therefore has 6 subunits:
α2ββ'σω (~480 kDa).
• When not in use RNA
polymerase binds to low affinity
sites to allow rapid exchange
for an active promoter site
when one opens. RNA
polymerase holoenzyme,
therefore, does not freely float
around in the cell when not in
use.
Model of bacterial RNA
The beta’ subunit is in
orange; beta is in green. Part
of one of the two alpha
subunits can be seen in light
blue; the omega subunit is in
grey. The DNA template and
non template strands
are shown, respectively, as
grey and pink ribbons. A Mg2+
ion at the active center is
shown as a grey sphere.
Numbers indicate positions in
the DNA sequence relative to
the transcription start site,
with positive (+) numbers in
the direction of transcription
and negative (-) numbers in
the opposite direction.
RNA polymerase in Eukaryotes
Eukaryotes have several types of RNAP,
characterized by the type of RNA they
synthesize, also they are more complex
structures (each with 2 large subunits &
10-14 smaller subunits) in comparison to
the prokaryotes. The best characterized
eukaryotic RNA polymerases are from the
yeast S. cerevisiae.
• RNA polymerase I synthesizes a pre-
rRNA 45S, which matures into 28S,
18S and 5.8S rRNAs which will form
the RNA polymerase II major RNA
sections of the ribosome.
• synthesizes precursors of mRNA and
most snRNA and microRNA.This is
the most studied type, its has a high
level of control over transcription
thus a range of transcription factors
are required for its binding to Essential Subunit Of Human RNA
promoters. Polymerases I, II and III
• RNA polymerase III synthesizes
tRNAs, rRNA 5S and other small
RNAsfound in the nucleus and
• Each eukaryotic RNA polymerase catalyzes
transcription of genes encoding different classes of
RNA. RNA polymerase I, located in the nucleolus,
transcribes genes encoding precursor rRNA (pre-
rRNA), which is processed into 28S, 5.8S,and 18S
rRNAs.
• RNA polymerase III transcribes genes encoding
tRNAs, 5S rRNA, and an array of small, stable RNAs,
including one involved in RNA splicing (U6) and the
RNA component of the signal-recognition particle (SRP)
involved in directing nascent proteins to the
endoplasmic reticulum.
• RNA polymerase II transcribes all protein-coding
genes; that is, it functions in production of mRNAs. RNA
polymerase II also produces four of the five small
nuclear RNAs that take part in RNA splicing.
• The two large subunits (RPB1 and
RPB2) of all three eukaryotic RNA
polymerases are related to each
other and are similar to the E. coli
subunits, respectively.
• The extensive similarity in the
structures of these core subunits in
RNA polymerases from various
sources indicates that this enzyme
arose early in evolution and was
largely conserved.
• Yeast RNA polymerases contain
four additional small subunits,
common to all the 3 types of RNAP
but not to the one in E.coli
• RNAP can initiate transcription at specific
DNA sequences known as promoters
&then produces an RNA chain which is
complementary to the template DNA
strand.
• The process of adding nucleotides to the
RNA strand is known as elongation;in
eukaryotes, RNAP can build chains as
long as 2.4 million nucleosides .RNAP will
preferentially release its RNA transcript at
specific DNA sequences encoded at the
end of genes known as terminators.

An electron-micrograph of DNA strands decorated by

hundreds of RNAP molecules too small to be resolved. Each
RNAP is transcribing an RNA strand which can be seen
branching off of the DNA. "Begin" indicates the 3' end of the
DNA, where RNAP initiates transcription; "End" indicates the
5' end, where the longer RNA molecules are almost completely
transcribed.
RNA polymerase action
In the process of transcription (by
any polymerase) there are three
main stages:
1. Initiation; the construction of the
RNA polymerase complex on the
gene's promoter with the help of
transcription factors.
2. Elongation; the actual
transcription of the majority of the
gene into a corresponding RNA
sequence.
3. Termination; the cessation of
RNA transcription and the
disassembly of the RNA
polymerase complex.
 Products of RNAP include:

• Messenger RNA (mRNA)—template for the synthesis of proteins by

ribosomes.
• Non-coding RNA or "RNA genes"—a broad class of genes that encode
RNA that is not translated into protein. The most prominent examples
of RNA genes are transfer RNA (tRNA) and ribosomal RNA (rRNA), both
of which are involved in the process of translation.
• Since the late 1990s, many new RNA genes have been found, and thus
RNA genes may play a much more significant role than previously
thought.
 Transfer RNA (tRNA)—transfers specific amino acids to growing
polypeptide chains at the ribosomal site of protein synthesis
during translation .
 Ribosomal RNA (rRNA)—a component of ribosomes .
 Micro RNA—regulates gene activity.
 Catalytic RNA (Ribozyme)—enzymatically active RNA molecules.

• RNAP accomplishes de novo synthesis. It is able to do this because

specific interactions with the initiating nucleotide hold RNAP rigidly in
place, facilitating chemical attack on the incoming nucleotide,thus
prefers to start transcripts with ATP (followed by GTP, UTP, and then
CTP).
• RNAP includes helicase activity, therefore no separate enzyme is
needed to unwind DNA.
RNA Polymerase Versus DNA
Polymerase
Many of the aspects of DNA and RNA Polymerases are
SIMILAR:
• Polymerization is ALWAYS 5’ to 3’
• Linkage is by phosphodiesterase bond formation
• Polymerization is driven by release and use of PPi
Several aspects of DNA and RNA Polymerases are
VERY DIFFERENT
• RNA polymerase only works on one strand
• RNA polymerase has it’s own helicase activity
• RNA polymerase does NOT need priming (primase)
• RNA does NOT stay bound following synthesis
• RNA polymerase does NOT proofread product (RNA)