RNA polymerase
201268
222325756
2008-06-28T19:03:26Z
DOI bot
6652755
Citation maintenance. Formatted: doi. You can [[WP:DOI|use this bot]] yourself! Please [[User:DOI_bot/bugs|report any bugs]].
[[Image:RNAP TEC small.jpg|thumb|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 an [[RNA]] copy of a [[DNA]] or RNA template. In [[cell (biology)|cell]]s, RNAP is needed for constructing RNA chains from DNA [[gene]]s, a process called [[Transcription (genetics)|transcription]]. RNA polymerase enzymes are essential to life and are found in all organisms and many [[virus]]es. In chemical terms, RNAP is a [[nucleotidyl transferase]] that [[polymerization|polymerizes]] [[ribonucleotide]]s at the [[3']] end of an RNA transcript.
==History==
RNAP was discovered independently by [[Sam Weiss]] and [[Jerard Hurwitz]] in 1960.<ref>{{cite journal|author=Jerard Hurwitz|year=2005|month=Dec|title=The Discovery of RNA Polymerase|journal=Journal of Biological Chemistry |volume=280|issue=52|pages=42477–85|pmid=16230341|doi=10.1074/jbc.X500006200}}</ref> 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<ref>[http://nobelprize.org/nobel_prizes/medicine/laureates/1959/ Nobel Prize 1959]</ref>, but instead turned out to be [[polynucleotide phosphorylase]].
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.<ref>[http://nobelprize.org/nobel_prizes/chemistry/laureates/2006/index.html Nobel Prize in Chemistry 2006]</ref>
==Control of transcription==
[[Image:Transcription label fromcommons.jpg|thumb|An [[electron-micrograph]] of [[DNA|DNA strands]] decorated by hundreds of RNAP molecules too small to be resolved. Each RNAP is transcribing an [[RNA|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 (genetics)|transcription]] affects patterns of [[gene expression]] and thereby allows a [[cell (biology)|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.<ref>{{cite journal |author=Akira Ishihama|title=Functional modulation of Escherichia coli RNA polymerase|year=2000|volume=54|pages=499-518|pmid=11018136 | doi = 10.1162/artl.2006.12.4.513 }}</ref>
RNAP can initiate [[Transcription (genetics)|transcription]] at specific DNA sequences known as [[promoter]]s. It then produces an RNA chain which is [[Complementarity (molecular biology)|complementary]] to the template DNA strand. The process of adding [[nucleotide]]s 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 [[Terminator (genetics)|terminators]].
Products of RNAP include:
*[[Messenger RNA]] (mRNA)—template for the [[Translation (biology)|synthesis of proteins]] by [[ribosome]]s.
*[[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 acid]]s to growing [[polypeptide]] chains at the ribosomal site of protein synthesis during [[translation (biology)|translation]]
**[[Ribosomal RNA]] (rRNA)—a component of ribosomes
**[[Micro RNA]]—regulates gene activity
**Catalytic RNA ([[Ribozyme]])—[[enzyme|enzymatically]] active RNA molecules
RNAP accomplishes [[de novo synthesis|''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 in prokaryotes involves the α subunit recognizing the upstream element (-40 to -70 base pairs) 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<sup>70</sup> is expressed under normal conditions and allows RNAP binding to house-keeping genes, while σ<sup>32</sup> 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 an 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 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 -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 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.
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 ([[Aspartic acid|asp]]) residues in the RNAP will hold onto Mg<sup>2+</sup> ions, which will in turn coordinate the phosphates of the ribonucleotides. The first Mg<sup>2+</sup> 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<sup>2+</sup> will hold onto the pyrophosphate of the NTP. The overall reaction equation is:
(NMP)<sub>n</sub> + NTP --> (NMP)<sub>n+1</sub> + PP<sub>i</sub>
===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 factor|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.<ref>{{cite journal |author=Farnham PJ|coauthors=Platt T.|title=Rho-independent termination: dyad symmetry in DNA causes RNA polymerase to pause during transcription in vitro|journal=Nucleic Acids Res.|year=1981 |month=Feb|volume=9|issue=3|pages=563-77|pmid=7012794 | doi = 10.1093/nar/9.3.563 }}</ref>
==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 [[Atomic mass unit|kDa]]):
*α<sub>2</sub>: 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. This subunit is not used on promoters without an UP element.
*[[rpoB|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<sub>2</sub>ββ'σω (~480 kDa). The structure of RNAP exhibits a groove with a length of 55 Å (5.5 [[nanometer|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 [[nucleotide]]s.
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'' and in most other prokaryotes 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==
[[Image:Alpha-Amanitin–RNA polymerase II complex 1K83.png|thumb|Structure of eukaryotic RNA polymerase II (light blue) in complex with α-amanitin (red), a strong poison found in death cap mushrooms that targets this vital enzyme]]
[[Eukaryote]]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]].<ref>{{cite journal|author=Grummt I.|title=Regulation of mammalian ribosomal gene transcription by RNA polymerase I.|journal=Prog Nucleic Acid Res Mol Biol.|year=1999|volume=62|pages=109–54|pmid=9932453|doi=10.1016/S0079-6603(08)60506-1}}</ref>
*[[RNA polymerase II]] synthesizes precursors of [[mRNA]]s and most [[snRNA]] and [[microRNA]]s.<ref>{{cite journal |author=Lee Y|coauthors=Kim M; Han J; Yeom KH; Lee S; Baek SH; Kim VN.|title=MicroRNA genes are transcribed by RNA polymerase II|journal=EMBO J.|year=2004|month=Oct|volume=23|issue=20|pages=4051-60|pmid=15372072 | doi = 10.1038/sj.emboj.7600385 }}</ref> This is the most studied type, and due to the high level of control required over transcription a range of [[transcription factor]]s are required for its binding to promoters.
*[[RNA polymerase III]] synthesizes [[tRNA]]s, [[rRNA]] 5S and other [[small RNA]]s found in the [[cell nucleus|nucleus]] and [[cytosol]].<ref>{{cite journal |author=Willis IM.|title=RNA polymerase III. Genes, factors and transcriptional specificity|journal=Eur J Biochem.|year=1993|month=Feb|volume=212|issue=1|pages=1-11|pmid=8444147 | doi = 10.1111/j.1432-1033.1993.tb17626.x }}</ref>
*[[RNA polymerase IV]] synthesizes [[Small interfering RNA|siRNA]] in plants.<ref>{{cite journal |author=Herr AJ, Jensen MB, Dalmay T, Baulcombe DC |title=RNA polymerase IV directs silencing of endogenous DNA |journal=Science |volume=308 |issue=5718 |pages=118–20 |year=2005 |pmid=15692015 |doi=10.1126/science.1106910}}</ref>
There are other RNA polymerase types in [[mitochondria]] and [[chloroplast]]s.
And there are [[RNA-dependent RNA polymerase]]s involved in [[RNA interference]].<ref>[http://www.ebi.ac.uk/interpro/IEntry?ac=IPR007855 InterPro: RNA-dependent RNA polymerase, eukaryotic-type], retrieved 6 April 2008.</ref>
==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.<ref>D Langer, J Hain, P Thuriaux and W Zillig (1995) Transcription in Archaea: Similarity to that in Eucarya PNAS '''92''' 5768-5772</ref>
==RNA polymerase in viruses==
[[Image:RNA pol.jpg|thumb|T7 RNA polymerase producing a mRNA (green) from a DNA template. The protein is shown as a purple ribbon. Image derived from [http://www.rcsb.org/pdb/explore/explore.do?structureId=1MSW PDB 1MSW].]]
Many [[virus]]es 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]].<ref>Hedtke ''et al.'' (1997) Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. Science '''227''' 809-811</ref> It is believed 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 [[DsRNA virus#Group V - negative-sense ssRNA viruses|negative strand RNA viruses]] and [[DsRNA virus#Group III - dsRNA viruses|dsRNA viruses]], both of which exist for a portion of their life cycle as double-stranded RNA. However, some [[DsRNA virus#Group IV - positive-sense ssRNA viruses|positive strand RNA viruses]], such as [[polio]], also contain these [[RNA dependent RNA polymerase]]s.<ref>Paul Ahlquist (2002) RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing. Science '''296''' 1270-1273</ref>
==RNA polymerase purification==
RNA polymerase can be isolated in the following ways:
*By a [[phosphocellulose column]].<ref>{{cite journal|author=Kelly JL|coauthors=Lehman IR.|title=Yeast mitochondrial RNA polymerase. Purification and properties of the catalytic subunit.
|journal=J Biol Chem.|year=1986|month=Aug|volume=261|issue=22|pages=10340–7
|pmid=3525543}}</ref>
*By [[glycerol gradient centrifugation]].<ref>{{cite journal
|author=Honda A et al
|title=Purification and molecular structure of RNA polymerase from influenza virus A/PR8.
|journal=J Biochem (Tokyo)|year=1990|month=Apr|volume=107|issue=4|pages=624–8
|pmid=2358436}}</ref>
*By a [[DNA column]].
*By an [[Ion exchange]] column.<ref>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</ref>
And also combinations of the above techniques.
==See also==
*[[DNA polymerase]]
*[[T7 RNA polymerase]]
*[[RNA polymerase I]]
*[[RNA polymerase II]]
*[[RNA polymerase III]]
*[[Alpha-amanitin]]
==References==
{{Reflist|2}}
==External links==
*[http://www.dnai.org DNAi] - DNA Interactive, including information and Flash clips on RNA Polymerase.
* {{MeshName|RNA+Polymerase}}
* {{EC number|2.7.7.6}}
{{Polymerases}}
[[Category:Gene expression]]
[[Category:RNA]]
[[Category:Enzymes]]
[[da:RNA polymerase]]
[[de:RNA-Polymerase]]
[[el:RNA πολυμεράση]]
[[es:ARN polimerasa]]
[[fr:ARN polymérase]]
[[it:RNA polimerasi (DNA-dipendente)]]
[[he:RNA פולימראז]]
[[nl:RNA-polymerase]]
[[ja:RNAポリメラーゼ]]
[[oc:ARN polimerasa]]
[[pl:Polimeraza RNA]]
[[pt:ARN-polimerase]]
[[ro:ARN-polimeraza]]
[[ru:РНК-полимераза]]
[[fi:RNA-polymeraasi]]
[[vi:RNA polymerase]]
[[tr:RNA polimeraz]]
[[uk:РНК-полімераза]]
[[zh:RNA聚合酶]]