RNA polymerase, also known as DNA-dependent RNA polymerase, is an
enzyme that produces primary transcript RNA. In cells, RNAP is necessary for
constructing RNA chains using DNA genes as templates, 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 ribonucleotides at the 3' end of an RNA
transcript. History
RNAP was discovered independently by Charles Loe, Audrey Stevens, and Jerard
Hurwitz in 1960. By this time, one half of the 1959 Nobel Prize in Medicine had
been awarded to Severo Ochoa for the discovery of what was believed to be
RNAP, but instead turned out to be polynucleotide phosphorylase.
The 2006 Nobel Prize in Chemistry was awarded to Roger D. Kornberg for
creating detailed molecular images of RNA polymerase during various stages of
the transcription process. Control of transcription
Control of the process of gene transcription affects patterns of gene
expression and, thereby, allows a cell to adapt to a changing environment,
perform specialized roles within an organism, and maintain basic metabolic
processes necessary for survival. Therefore, it is hardly surprising that
the activity of RNAP is both long and complex and highly regulated. In
Escherichia coli bacteria, more than 100 transcription factors have been
identified, which modify the activity of RNAP.
RNAP can initiate transcription at specific DNA sequences known as
promoters. It 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
nucleotides. RNAP will preferentially release its RNA transcript at specific
DNA sequences encoded at the end of genes, which are known as terminators.
Products of RNAP include: Messenger RNA—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 and ribosomal RNA, both of
which are involved in the process of translation. However, 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—transfers specific amino acids to growing polypeptide chains at
the ribosomal site of protein synthesis during translation
Ribosomal RNA—a component of ribosomes Micro RNA—regulates gene activity
Catalytic RNA—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. Such specific
interactions explain why RNAP prefers to start transcripts with ATP. In contrast
to DNA polymerase, RNAP includes helicase activity, therefore no separate
enzyme is needed to unwind DNA. Action
RNA polymerase binding in bacteria involves the sigma factor recognizing
the core promoter region containing the -35 and -10 elements and also, at some
promoters, the α subunit C-terminal domain recognizing promoter upstream
elements. There are multiple interchangeable sigma factors, each of
which recognizes a distinct set of promoters. For example, in E. coli, σ70
is expressed under normal conditions and recognizes promoters for genes required
under normal conditions, while σ32 recognizes promoters for genes required
at high temperatures. After binding to the DNA, the RNA
polymerase switches from a closed complex to an open complex. This change
involves the separation of the DNA strands to form an unwound section of
DNA of approximately 13 bp, referred to as the transcription bubble.
Ribonucleotides are base-paired to the template DNA strand, according to
Watson-Crick base-pairing interactions. Supercoiling plays an important part in
polymerase activity because of the unwinding and rewinding of DNA. Because
regions of DNA in front of RNAP are unwound, there are compensatory positive
supercoils. Regions behind RNAP are rewound and negative supercoils are
present. As noted above, RNA polymerase makes
contacts with the promoter region. However these stabilizing contacts
inhibit the enzyme's ability to access DNA further downstream and thus the
synthesis of the full-length product. Once the open complex is stabilized, RNA
polymerase synthesizes an RNA strand to establish a DNA-RNA heteroduplex at the
active center, which stabilizes the elongation complex. In order to
accomplish RNA synthesis, RNA polymerase must maintain promoter contacts while
unwinding more downstream DNA for synthesis, "scrunching" more downstream
DNA into the initiation complex. During the promoter escape transition, RNA
polymerase is considered a "stressed intermediate." Thermodynamically the
stress accumulates from the DNA-unwinding and DNA-compaction
activities. Once the DNA-RNA heteroduplex is long enough, RNA
polymerase releases its upstream contacts and effectively achieves the
promoter escape transition into the elongation phase. However, promoter
escape is not the only outcome. RNA polymerase can also relieve the stress
by releasing its downstream contacts, arresting transcription. The paused
transcribing complex has two options: release the nascent transcript and begin
anew at the promoter or reestablish a new 3'OH on the nascent transcript at
the active site via RNA polymerase's catalytic activity and recommence DNA
scrunching to achieve promoter escape. Scientists have coined the term
"abortive initiation" to explain the unproductive cycling of RNA polymerase
before the promoter escape transition. The extent of abortive initiation
depends on the presence of transcription factors and the strength of the promoter
contacts. = Elongation=
Transcription elongation involves the further addition of ribonucleotides and
the change of the open complex to the transcriptional complex. RNAP cannot
start forming full length transcripts because of its strong binding to the
promoter. Transcription at this stage primarily results in short RNA fragments
of around 9 bp in a process known as abortive transcription. Once the RNAP
starts forming longer transcripts it clears the promoter. At this point, the
contacts with the -10 and -35 elements are disrupted, and the σ factor falls
off RNAP. This allows the rest of the RNAP complex to move forward, as the σ
factor held the RNAP complex in place. The 17-bp transcriptional complex has an
8-bp DNA-RNA hybrid, that is, 8 base-pairs involve the RNA transcript
bound to the DNA template strand. As transcription progresses,
ribonucleotides are added to the 3' end of the RNA transcript and the RNAP
complex moves along the DNA. Although RNAP does not seem to have the
3'exonuclease activity that characterizes the proofreading activity
found in DNA polymerase, there is evidence of that RNAP will halt at
mismatched base-pairs and correct it. Aspartyl residues in the RNAP will hold
on to Mg2+ ions, which will, in turn, coordinate the phosphates of the
ribonucleotides. The first Mg2+ will hold on to the α-phosphate of the NTP to
be added. This allows the nucleophilic attack of the 3'OH from the RNA
transcript, adding another NTP to the chain. The second Mg2+ will hold on to
the pyrophosphate of the NTP. The overall reaction equation is:
(NMP)n + NTP -->n+1 + PPi = Termination=
In prokaryotes, termination of RNA transcription can be rho-independent or
rho-dependent: Rho-independent transcription
termination is the termination of transcription without the aid of the rho
protein. Transcription of a palindromic region of DNA causes the formation of a
"hairpin" structure from the RNA transcription looping and binding upon
itself. This hairpin structure is often rich in G-C base-pairs, making it more
stable than the DNA-RNA hybrid itself. As a result, the 8 bp DNA-RNA hybrid in
the transcription complex shifts to a 4 bp hybrid. These last 4 base pairs are
weak A-U base pairs, and the entire RNA transcript will fall off the DNA.
In bacteria In bacteria, the same enzyme catalyzes
the synthesis of mRNA and ncRNA. RNAP is a large molecule. The core
enzyme has five subunits: β': The β' subunit is the largest
subunit, and is encoded by the rpoC gene. The β' subunit contains part of
the active center responsible for RNA synthesis and contains some of the
determinants for non-sequence-specific interactions with DNA and nascent RNA.
β: The β subunit is the second-largest subunit, and is encoded by the rpoB
gene. The β subunit contains the rest of the active center responsible for RNA
synthesis and contains the rest of the determinants for non-sequence-specific
interactions with DNA and nascent RNA. αI and αII: The α subunit is the
third-largest subunit and is present in two copies per molecule of RNAP, αI and
αII. Each α subunit contains two domains: αNTD and αCTD. αNTD contains
determinants for assembly of RNAP. αCTD contains determinants for interaction
with promoter DNA, making non-sequence-non-specific interactions
at most promoters and sequence-specific interactions at
upstream-element-containing promoters, and contains determinants for
interactions with regulatory factors. ω: The ω subunit is the smallest
subunit. The ω subunit facilitates assembly of RNAP and stabilizes
assembled RNAP. In order to bind promoters, RNAP core
associates with the transcription initiation factor sigma to form RNA
polymerase holoenzyme. Sigma reduces the affinity of RNAP for nonspecific DNA
while increasing specificity for promoters, allowing transcription to
initiate at correct sites. The complete holoenzyme therefore has 6 subunits:
β'βαI and αIIωσ. In eukaryotes
Eukaryotes have multiple types of nuclear RNAP, each responsible for
synthesis of a distinct subset of RNA. All are structurally and mechanistically
related to each other and to bacterial RNAP:
RNA polymerase I synthesizes a pre-rRNA 45S, which matures into 28S, 18S and
5.8S rRNAs which will form the major RNA sections of the ribosome.
RNA polymerase II synthesizes precursors of mRNAs and most snRNA and microRNAs.
This is the most studied type, and, due to the high level of control required
over transcription, a range of transcription factors are required for
its binding to promoters. RNA polymerase III synthesizes tRNAs,
rRNA 5S and other small RNAs found in the nucleus and cytosol.
RNA polymerase IV synthesizes siRNA in plants.
RNA polymerase V synthesizes RNAs involved in siRNA-directed
heterochromatin formation in plants. Eukaryotic chloroplasts contain an RNAP
very highly structurally and mechanistically similar to bacterial
RNAP. Eukaryotic chloroplasts also contain a
second, structurally and mechanistically unrelated, RNAP.
Eukaryotic mitochondria contain a structurally and mechanistically
unrelated RNAP. Given that DNA and RNA polymerases both
carry out template-dependent nucleotide polymerization, it might be expected
that the two types of enzymes would be structurally related. However, x-ray
crystallographic studies of both types of enzymes reveal that, other than
containing a critical Mg2+ ion at the catalytic site, they are virtually
unrelated to each other; indeed template-dependent nucleotide
polymerizing enzymes seem to have arisen independently twice during the early
evolution of cells. One lineage led to the modern DNA Polymerases and reverse
transcriptases, as well as to a few single-subunit RNA polymerases from
viruses. The other lineage formed all of the modern cellular RNA polymerases.
In archaea Archaea have a single type of RNAP,
responsible for the synthesis of all RNA. Archaeal RNAP is structurally and
mechanistically similar to bacterial RNAP and eukaryotic nuclear RNAP I-V,
and is especially closely structurally and mechanistically related to
eukaryotic nuclear RNAP II. The history of the discovery of the archaeal RNA
polymerase is quite recent. The first analysis of the RNAP of an archaeon was
performed in 1971, when the RNAP from the extreme halophile Halobacterium
cutirubrum was isolated and purified. Crystal structures of RNAPs from
Sulfolobus solfataricus and Sulfolobus shibatae set the total number of
identified archaeal subunits at thirteen.
In viruses Orthopoxviruses synthesize RNA using a
virally encoded RNAP that is structurally and mechanistically related
to bacterial RNAP, archaeal RNAP, and eukaryotic nuclear RNAP I-V. Most other
viruses that synthesize RNA using a virally encoded RNAP use an RNAP that is
not structurally and mechanistically related to bacterial RNAP, archaeal
RNAP, and eukaryotic nuclear RNAP I-V. Many viruses use a single-subunit
DNA-dependent RNAP that is structurally and mechanistically related to the
single-subunit RNAP of eukaryotic chloroplasts and mitochondria and, more
distantly, to DNA polymerases and reverse transcriptases. Perhaps the most
widely studied such single-subunit RNAP is bacteriophage T7 RNA polymerase.
Other viruses use a RNA-dependent RNAP. This occurs in negative strand RNA
viruses and dsRNA viruses, both of which exist for a portion of their life cycle
as double-stranded RNA. However, some positive strand RNA viruses, such as
poliovirus, also contain RNA-dependent RNAP.
Purification RNA polymerase can be isolated in the
following ways: By a phosphocellulose column.
By glycerol gradient centrifugation. By a DNA column.
By an ion chromatography column. And also combinations of the above
techniques. See also
Alpha-amanitin DNA polymerase
RNA polymerase I RNA polymerase II
RNA polymerase III T7 RNA polymerase
Transcription Transcription Activators in Eukaryotes
References External links
DNAi - DNA Interactive, including information and Flash clips on RNA
Polymerase. RNA Polymerase at the US National
Library of Medicine Medical Subject Headings
EC 2.7.7.6 RNA Polymerase - Synthesis RNA from DNA
Template 3D macromolecular structures of RNA
Polymerase from the EM Data Bank(EMDB)