RNA polymerase

In molecular biology, RNA polymerase, or more specifically DNA-directed/dependent RNA polymerase, 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 ; or non-coding. Examples of four functional types of RNA genes are:
; Transfer RNA : Transfers specific amino acids to growing polypeptide chains at the ribosomal site of protein synthesis during translation;
; Ribosomal RNA : Incorporates into ribosomes;
; Micro RNA : Regulates gene activity; and, RNA silencing
; Catalytic RNA : 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 or only consist of one subunit, each representing an independent lineage. The former is found in bacteria, archaea, and eukaryotes alike, sharing a similar core structure and mechanism. The latter is found in phages as well as eukaryotic chloroplasts and mitochondria, and is related to modern DNA polymerases. Eukaryotic and archaeal RNAPs have more subunits than bacterial ones do, and are controlled differently.
Bacteria and archaea only have one RNA polymerase. Eukaryotes have multiple types of nuclear RNAP, each responsible for synthesis of a distinct subset of RNA:

Structure

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 subunits of 36 kDa, a beta subunit of 150 kDa, a beta prime subunit of 155 kDa, and a small omega subunit. A sigma 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.
All RNAPs contain metal cofactors, in particular zinc and magnesium cations which aid in the transcription process.

Function

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 long, 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

Initiation

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. 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.

Promoter escape

RNA polymerase then starts to synthesize the initial DNA-RNA heteroduplex, with ribonucleotides base-paired to the template DNA strand according to Watson-Crick base-pairing interactions. 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. In order to continue RNA synthesis, RNA polymerase must escape the promoter. It 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. 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: 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. 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.

Elongation

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. The characteristic elongation rates in prokaryotes and eukaryotes are about 10–100 nts/sec.
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:

Fidelity

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.