RNA polymerase

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

Transfer RNA (tRNA)

Transfers specific

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.

Structure

T. aquaticus RNA polymerase core (PDB: 1HQM​).

Yeast RNA polymerase II core (PDB: 1WCM​).

Homologous subunits are colored the same:^[1]

  orange: α1/RPB3,

  yellow: α2/RPB11,

  wheat: β/RPB2,

  red: β′/RPB1,

  pink: ω/RPB6.

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.^[3][4]

In most

All RNAPs contain metal cofactors, in particular zinc and magnesium cations which aid in the transcription process.^[9][10]

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.^[11]

RNAP can initiate transcription at specific DNA sequences known as

• Messenger RNA (mRNA)—template for the synthesis of proteins by ribosomes.
• 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.
  • 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.

Action

Initiation

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.^[12] 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."^[13][14]

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 "

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.^[16]

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.^[17] 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.^[18]

Aspartyl (asp) 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.^[19] The overall reaction equation is:

(NMP)_n + NTP → (NMP)_n+1 + PP_i

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.^[20]

The overall error rate is around 10⁻⁴ to 10⁻⁶.^[21]

Termination

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.^[22] 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.^[23]

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.^[24]

Other organisms

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.^[2] The other multi-subunit RNAP lineage formed all of the modern cellular RNA polymerases.^[25][1]

Bacteria

In

β′

The β′ subunit is the largest subunit, and is encoded by the rpoC gene.[27] 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.^[28]

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 (α^I and α^II)

Two copies of the α subunit, being the third-largest subunit, are present in a molecule of RNAP: α^I and α^II (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.^[29]

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′βα^I and α^IIωσ (~450 kDa).

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:

Eukaryotic chloroplasts contain an RNAP very highly similar to bacterial RNAP ("plastid-encoded polymerase, PEP"). They use sigma factors encoded in the nuclear genome.^[35]

Chloroplast also contain a second, structurally and mechanistically unrelated, single-subunit RNAP ("nucleus-encoded polymerase, NEP"). Eukaryotic

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.^[8][36] 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

Archaeal RNAP subunit previously used an "RpoX" nomenclature where each subunit is assigned a letter in a way unrelated to any other systems.[1] In 2009, a new nomenclature based on Eukaryotic Pol II subunit "Rpb" numbering was proposed.^[8]

Viruses

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.^[2]

RNAP was discovered independently by Charles Loe, Audrey Stevens, and Jerard Hurwitz in 1960.^[46] 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,^[47] but instead turned out to be polynucleotide phosphorylase.

Purification

RNA polymerase can be isolated in the following ways:

• By a phosphocellulose column.^[48]
• By glycerol gradient centrifugation.^[49]
• By a DNA column.
• By an ion chromatography column.^[50]

And also combinations of the above techniques.

See also

• Alpha-amanitin
• Primase

References

External links

Wikimedia Commons has media related to RNA polymerase.

• DNAi – DNA Interactive, including information and Flash clips on RNA Polymerase.
• RNA+Polymerase at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
• EC 2.7.7.6
• RNA Polymerase – Synthesis RNA from DNA Template

(Wayback Machine copy)

• 3D macromolecular structures of RNA Polymerase from the EM Data Bank(EMDB)

This article incorporates text from the public domain Pfam and InterPro: IPR011773