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Enzyme that synthesizes RNA from DNA

In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the double stranded DNA so that one strand of the exposed nucleotides can be used as a template for the synthesis of RNA, a process called transcription. A transcription factor and its associated transcription mediator complex must be attached to a DNA binding site called a promoter region before RNAP can initiate the DNA unwinding at that position. RNAP not only initiates RNA transcription, it also guides the nucleotides into position, facilitates attachment and elongation, has intrinsic proofreading and replacement capabilities, and termination recognition capability. In eukaryotes, RNAP can build chains as long as 2.4 million nucleotides. RNAP produces RNA that, functionally, is either for protein coding, i. e. messenger RNA (mRNA); or non coding (so called "RNA genes"). Examples of four functional types of RNA genes are:
Transfer RNA (tRNA) Transfers specific amino acids to growing polypeptide chains at the ribosomal site of protein synthesis during translation;
Ribosomal RNA (rRNA) Incorporates into ribosomes;
Micro RNA (miRNA) Regulates gene activity; and, RNA silencing
Catalytic RNA (ribozyme) Functions as an enzymatically active RNA molecule. RNA polymerase is essential to life, and is found in all living organisms and many viruses. Depending on the organism, a RNA polymerase can be a protein complex (multi subunit RNAP) or only consist of one subunit (single subunit RNAP, ssRNAP), each representing an independent lineage. The former is found in bacteria, archaea, and eukaryotes alike, sharing a similar core structure and mechanism. 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. In most prokaryotes, a single RNA polymerase species transcribes all types of RNA. RNA polymerase "core" from E. coli consists of five subunits: two alpha (α) subunits of 36 kDa, a beta (β) subunit of 150 kDa, a beta prime subunit (β′) of 155 kDa, and a small omega (ω) subunit. A sigma (σ) factor binds to the core, forming the holoenzyme. After transcription starts, the factor can unbind and let the core enzyme proceed with its work. The core RNA polymerase complex forms a "crab claw" or "clamp jaw" structure with an internal channel running along the full length. Eukaryotic and archaeal RNA polymerases have a similar core structure and work in a similar manner, although they have many extra subunits.

RNA polymerase binding in bacteria involves the sigma factor recognizing the core promoter region containing the −35 and −10 elements (located before the beginning of sequence to be transcribed) 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 ("housekeeping genes"), while σ32 recognizes promoters for genes required at high temperatures ("heat shock genes"). In archaea and eukaryotes, the functions of the bacterial general transcription factor sigma are performed by multiple general transcription factors that work together. The RNA polymerase promoter closed complex is usually referred to as the "transcription preinitiation complex." 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". 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. 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 (~10 bp), RNA polymerase releases its upstream contacts and effectively achieves the promoter escape transition into the elongation phase. The heteroduplex at the active center stabilizes the elongation complex. 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: (1) release the nascent transcript and begin anew at the promoter or (2) 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. Abortive initiation, the unproductive cycling of RNA polymerase before the promoter escape transition, results in short RNA fragments of around 9 bp in a process known as abortive transcription. The extent of abortive initiation depends on the presence of transcription factors and the strength of the promoter contacts.

Unlike the proofreading mechanisms of DNA polymerase those of RNAP have only recently been investigated. Proofreading begins with separation of the mis incorporated nucleotide from the DNA template. This pauses transcription. The polymerase then backtracks by one position and cleaves the dinucleotide that contains the mismatched nucleotide. In the RNA polymerase this occurs at the same active site used for polymerization and is therefore markedly different from the DNA polymerase where proofreading occurs at a distinct nuclease active site. The overall error rate is around 10−4 to 10−6.

In bacteria, termination of RNA transcription can be rho dependent or rho independent. The former relies on the rho factor, which destabilizes the DNA RNA heteroduplex and causes RNA release. The latter, also known as intrinsic termination, relies on a palindromic region of DNA. Transcribing the region 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. Transcription termination in eukaryotes is less well understood than in bacteria, but involves cleavage of the new transcript followed by template independent addition of adenines at its new 3′ end, in a process called polyadenylation.

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 (ssRNAP) from phages and organelles. 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. It is split into two subunits in Cyanobacteria and chloroplasts. 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. α (αI and αII) Two copies of the α subunit, being the third largest subunit, are present in a molecule of RNAP: αI and αII (one and two). Each α subunit contains two domains: αNTD (N terminal domain) and αCTD (C terminal domain). αNTD contains determinants for assembly of RNAP. αCTD (C terminal domain) 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 (σ) 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 (~450 kDa).

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:

Eukaryotic chloroplasts contain an RNAP very highly similar to bacterial RNAP ("plastid encoded polymerase, PEP"). They use sigma factors encoded in the nuclear genome. Chloroplast also contain a second, structurally and mechanistically unrelated, single subunit RNAP ("nucleus encoded polymerase, NEP"). Eukaryotic mitochondria use POLRMT (human), a nucleus encoded single subunit RNAP. Such phage like polymerases are referred to as RpoT in plants. 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. Archaea has the subunit corresponding to Eukaryotic Rpb1 split into two. There is no homolog to eukaryotic Rpb9 (POLR2I) in the S. shibatae complex, although TFS (TFIIS homolog) has been proposed as one based on similarity. There is an additional subunit dubbed Rpo13; together with Rpo5 it occupies a space filled by an insertion found in bacterial β′ subunits (1,377–1,420 in Taq). This domain, in either case, serves a structural function. Archaeal RNAP subunit previously used an "RpoX" nomenclature where each subunit is assigned a letter in a way unrelated to any other systems. Exactly which RNAP they are most similar to is a topic of debate. Most other viruses that synthesize RNA use unrelated mechanics. Many viruses use a single subunit DNA dependent RNAP (ssRNAP) that is structurally and mechanistically related to the single subunit RNAP of eukaryotic chloroplasts (RpoT) and mitochondria (POLRMT) and, more distantly, to DNA polymerases and reverse transcriptases. Perhaps the most widely studied such single subunit RNAP is bacteriophage T7 RNA polymerase. ssRNAPs cannot proofread. Other viruses use an RNA dependent RNAP (an RNAP that employs 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 poliovirus, also contain RNA dependent RNAP.

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.