RNAP from ''T. aquaticus'' pictured during elongation. Portions of the enzyme were made transparent so as to make the path of RNA and DNA more clear. The magnesium ion (yellow) is located at the enzyme active site.
'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 ribonucleotides at the
3' end of an RNA transcript.
History
RNAP was discovered independently by
Sam Weiss and
Jerard Hurwitz in
1960.
[1] By this time the 1959
Nobel Prize in Medicine had been awarded to
Severo Ochoa and
Arthur Kornberg for the discovery of what was believed to be RNAP
[2], but instead turned out to be a
ribonuclease.
The 2006 Nobel Prize in Chemistry was awarded to
Roger Kornberg for creating detailed molecular images of RNA polymerase during various stages of the transcription process.
[3]
Control of transcription
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.
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 complex and highly regulated. In ''
Escherichia coli'' bacteria, more than 100 factors have been identified which modify the activity of RNAP.
[4]
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
nucleosides (the full length of the
dystrophin gene). RNAP will preferentially release its RNA transcript at specific DNA sequences encoded at the end of genes known as
terminators.
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. 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 (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. Such specific interactions explain why RNAP prefers to start transcripts with ATP (followed by GTP, UTP, and then CTP). In contrast to
DNA polymerase, RNAP includes
helicase activity, therefore no separate enzyme is needed to unwind DNA.
RNA polymerase action
Binding and initiation
RNA Polymerase binding involves the α subunit recognizing the upstream element (-40 to -70) in DNA, as well as the σ factor recognizing the -10 to -35 region. There are numerous σ factors that regulate gene expression. For example, σ
70 is expressed under normal conditions and allows RNAP binding to house-keeping genes, while σ
32 elicits RNAP binding to heat-shock genes.
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 a unwound section of DNA of approximately 13bp. 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 is compensatory positive supercoils. Regions behind RNAP are rewound and negative supercoils are present.
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 promoter. Transcription starts and mostly ends with short RNA fragments of around 9bp and the process is known as abortive transcription.Once the RNAP starts forming longer transcripts they gets releived from the promoter and this is known as promoter clearence.At this point, the -10 to -35 promoter region is 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 17bp transcriptional complex has an 8bp 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.
The addition of ribonucleotides to the RNA transcript has a very similar mechanism to DNA polymerization - it is believed that these polymerases are evolutionarily related. Aspartyl (
asp) residues in the RNAP will hold onto Mg
2+ ions, which will in turn coordinate the phosphates of the ribonucleotides. The first Mg
2+ will hold onto the α-phosphate of the NTP to be added. This allows the nucleophilic attack of the 3'OH from the RNA transcript, adding an additional NTP to the chain. The second Mg
2+ will hold onto the pyrophosphate of the NTP. The overall reaction equation is:
(NMP)
n + NTP --> (NMP)
n+1 + PP
i
Termination
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 8bp DNA-RNA hybrid in the transcription complex shifts to a 4bp hybrid. Coincidentally, these last 4 base-pairs are weak A-U base-pairs, and the entire RNA transcript will fall off.
[5]
RNA polymerase in bacteria
In
bacteria, the same enzyme catalyzes the synthesis of
mRNA and
ncRNA.
RNAP is a relatively large molecule. The core enzyme has 5 subunits (~400
kDa):
★ α
2: the two α subunits assemble the enzyme and recognize regulatory factors. Each subunit has two domains: αCTD (C-Terminal domain) binds the UP element of the extended promoter, and αNTD (N-terminal domain) binds the rest of the polymerase.
★
β: this has the polymerase activity (catalyzes the synthesis of RNA) which includes chain initiation and elongation.
★ β': binds to DNA (nonspecifically).
★ ω: restores denatured RNA polymerase to its functional form in vitro. It has been observed to offer a protective/chaperone function to the β' subunit in ''
Mycobacterium smegmatis''. Now known to promote assembly.
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. That way, transcription is initiated at the right region. The complete
holoenzyme therefore has 6 subunits: α
2ββ'σω (~480 kDa). The structure of RNAP exhibits a groove with a length of 55 Å (5.5
nm) and a diameter of 25 Å (2.5 nm). This groove fits well the 20 Å (2 nm) double strand of DNA. The 55 Å (5.5 nm) length can accept 16
nucleotides.
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.
Transcriptional cofactors
There are a number of proteins which can bind to RNAP and modify its behavior. For instance, greA and greB from ''E. coli'' can enhance the ability of RNAP to cleave the RNA template near the growing end of the chain. This cleavage can rescue a stalled polymerase molecule, and is likely involved in proofreading the occasional mistakes made by RNAP. A separate cofactor, Mfd, is involved in
transcription-coupled repair, the process in which RNAP recognizes damaged bases in the DNA template and recruits enzymes to restore the DNA. Other cofactors are known to play regulatory roles, i.e. they help RNAP choose whether or not to express certain genes.
RNA polymerase in eukaryotes
Essential Subunit Of Human RNA Polymerases I, II and III
s have several types of RNAP, characterized by the type of RNA they synthesize:
★
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.
[6]
★
RNA polymerase II synthesizes precursors of
mRNAs and most
snRNA and
microRNAs.
[7] 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.
[8]
There are other RNA polymerase types in
mitochondria and
chloroplasts.
RNA polymerase in archaea
Archaea have a single RNAP that is closely related to the three main eukaryotic polymerases. Thus, it has been speculated that the archaeal polymerase resembles the ancestor of the specialized eukaryotic polymerases.
[9]
RNA polymerase in viruses
T7 RNA polymerase producing a mRNA (green) from a DNA template. The protein is shown as a purple ribbon. Image derived from
PDB 1MSW.
Many
viruses also encode for RNAP. Perhaps the most widely studied viral RNAP is found in
bacteriophage T7. This single-subunit RNAP is related to that found in mitochondria and chloroplasts, and shares considerable homology to
DNA polymerase.
[10] It is believed by many that most viral polymerases therefore evolved from DNA polymerase and are not directly related to the multi-subunit polymerases described above.
The viral polymerases are diverse, and include some forms which can use RNA as a template instead of DNA. 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
polio, also contain these
RNA dependent RNA polymerases.
[11]
Functional domains
C-terminal domain of RNA polymerase
Transcription Initiation
The carboxy-terminal domain (CTD) of
RNA polymerase II is that portion of the polymerase which is involved in the initiation of
DNA transcription. The CTD typically consists of up to 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser
[12]. The transcription factor TFIIH is a kinase and will hyperphosphorylate the CTD of RNAP, and in doing so, causes the RNAP complex to move away from the initiation site.
5'Capping
The carboxy-terminal domain is also the binding site of the cap-synthesizing and cap-binding complex. In eukaryotes, after transcription of the 5' end of an RNA transcript, the cap-synthesizing complex on the CTD will remove the gamma-phosphate from the 5'phosphate and attach a GMP, forming a 5',5'-triphosphate linkage. The synthesizing complex falls off and the cap then binds to the cap-binding complex (CBC), which is bound to the CTD.
The 5'cap of eukaryotic RNA transcripts is important for binding of the RNA transcript to the ribosome during transcription, to the CTD of RNAP, and prevents RNA degradation.
Spliceosome
The carboxy-terminal domain is also the binding site for
spliceosome factors that are part of
RNA splicing. These allow for the splicing and removal of introns (in the form of a lariat structure) during RNA transcription.
Mutation in the CTD
Major studies have been carried out in which knockout of particular
amino acids was achieved in the CTD. The results indicate that RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of genes ''in vivo'', and the lack of response to induction maps to the upstream activating sequences of these genes.
RNA polymerase purification
RNA polymerase can be isolated in the following ways:
★ By a
phosphocellulose column.
[13]
★ By
glycerol gradient centrifugation.
[14]
★ By a
DNA column.
★ By an
Ion exchange column.
[15]
And also combinations of the above techniques.
See also
★
DNA polymerase
★
T7 RNA polymerase
★
Alpha-amanitin
References
1. The Discovery of RNA Polymerase, Jerard Hurwitz, , , Journal of Biological Chemistry, 2005
2. Nobel Prize 1959
3. Nobel Prize in Chemistry 2006
4. Functional modulation of Escherichia coli RNA polymerase, Akira Ishihama, , , , 2000
5. Rho-independent termination: dyad symmetry in DNA causes RNA polymerase to pause during transcription in vitro, Farnham PJ, , , Nucleic Acids Res., 1981
6. Regulation of mammalian ribosomal gene transcription by RNA polymerase I., Grummt I., , , Prog Nucleic Acid Res Mol Biol., 1999
7. MicroRNA genes are transcribed by RNA polymerase II, Lee Y, , , EMBO J., 2004
8. RNA polymerase III. Genes, factors and transcriptional specificity, Willis IM., , , Eur J Biochem., 1993
9. D Langer, J Hain, P Thuriaux and W Zillig (1995) Transcription in Archaea: Similarity to that in Eucarya PNAS '92' 5768-5772
10. Hedtke ''et al.'' (1997) Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. Science '227' 809-811
11. Paul Ahlquist (2002) RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing. Science '296' 1270-1273
12. Recognition of RNA polymerase II carboxy-terminal domain by 3'-RNA-processing factors, Anton Meinhart1, , , Nature, 2004
13. Yeast mitochondrial RNA polymerase. Purification and properties of the catalytic subunit., Kelly JL, , , J Biol Chem., 1986
14. Purification and molecular structure of RNA polymerase from influenza virus A/PR8., Honda A et al, , , J Biochem (Tokyo), 1990
15. Hager ''et al.'' (1990) Use of Mono Q High-Resolution Ion-Exchange Chromatography To Obtain Highly Pure and Active Escherichia coli RNA Polymerase Biochemistry '29' 7890-7894
External links
★
DNAi - DNA Interactive, including information and Flash clips on RNA Polymerase.
★
★
العربية
中国
Français
Deutsch
Ελληνική
हिन्दी
Italiano
日本語
Português
Русский
Español
