RNA Polymerase
RNA polymerase (RNA pol) is one of the most important enzymes in organic life as it is an essential block in the gene expression building. All kinds of RNA molecules are transcribed by RNA pols in any organism before they mature and get involved in life support processes — protein synthesis (mRNA, rRNA, tRNA), and various regulatory reactions (siRNA, hnRNA, etc.).
RNA polymerase and transcription enzymes
Discovery
RNA polymerase was discovered in 1959, independently in four American laboratories by Audrey Stevens, Samuel Weiss, James Bonner and Jerard Hurwitz. Three different sources were used: bacteria (E. coli), plant (peas) and animal (rat liver) to prepare reaction mixes, where the RNA synthesis in presence of rNTPs and DNA, was detected. Further Hurwitz, John Furth and Monika Anders purified the enzyme from E. coli extracts. The isolated RNA polymerase created RNA by combining adenine, uracil, guanine, and cytosine ribonucleoside triphosphates in the presence of DNA and either Mn2+ or Mg2+ [1].
RNA polymerase and transcription enzymes
Discovery
RNA polymerase was discovered in 1959, independently in four American laboratories by Audrey Stevens, Samuel Weiss, James Bonner and Jerard Hurwitz. Three different sources were used: bacteria (E. coli), plant (peas) and animal (rat liver) to prepare reaction mixes, where the RNA synthesis in presence of rNTPs and DNA, was detected. Further Hurwitz, John Furth and Monika Anders purified the enzyme from E. coli extracts. The isolated RNA polymerase created RNA by combining adenine, uracil, guanine, and cytosine ribonucleoside triphosphates in the presence of DNA and either Mn2+ or Mg2+ [1].
RNA polymerase types
RNA polymerases may be classified on the basis of organism class, kind of template usage, type of RNA transcribed, structure of enzyme and mechanism evolved in transcription.
DNA-dependent RNA polymerases (DDRP)
Prokaryotic
Prokaryotes RNA polymerase is a holoenzyme and consists of six subunits involving in different stages of RNA transcription (binding to the template, elongation, interaction with promoter, stabilization of the enzymatic complex, etc.). All types of bacteria or archaea RNAs are transcribed by the same enzyme in one organism.
Eukaryotic
For nowadays, it was discovered several types of eukaryotic RNA polymerase (I – V) synthesizing different RNA molecules in nuclei of plants, animals and fungi. All these RNA pols are structurally similar to prokaryotic prototype. Another RNA pols are responsible for RNA synthesis in chloroplasts and mitochondria — PEP [2], NEP [3] (chloroplasts) and POLRMT [4] (mitochondria): the first enzyme is similar to prokaryotic, and the two latter are closer to phage enzymes and consist of single subunit.
Viral
Viral DNA-dependent RNA polymerases are represented by two type enzymes. Large DNA viruses, such as Orthopoxvirus, possess multisubunit RNA pols that are similar to RNA polymerase II of eukaryotes and their activity in cells is supported by host transcriptional factors. Other DNA viruses, including phages, encode a single subunit RNA pol that is closer to DNA polymerases and reverse transcriptase than to multisubunit RNA pols [5]
DNA-dependent RNA polymerases (DDRP)
Prokaryotic
Prokaryotes RNA polymerase is a holoenzyme and consists of six subunits involving in different stages of RNA transcription (binding to the template, elongation, interaction with promoter, stabilization of the enzymatic complex, etc.). All types of bacteria or archaea RNAs are transcribed by the same enzyme in one organism.
Eukaryotic
For nowadays, it was discovered several types of eukaryotic RNA polymerase (I – V) synthesizing different RNA molecules in nuclei of plants, animals and fungi. All these RNA pols are structurally similar to prokaryotic prototype. Another RNA pols are responsible for RNA synthesis in chloroplasts and mitochondria — PEP [2], NEP [3] (chloroplasts) and POLRMT [4] (mitochondria): the first enzyme is similar to prokaryotic, and the two latter are closer to phage enzymes and consist of single subunit.
Viral
Viral DNA-dependent RNA polymerases are represented by two type enzymes. Large DNA viruses, such as Orthopoxvirus, possess multisubunit RNA pols that are similar to RNA polymerase II of eukaryotes and their activity in cells is supported by host transcriptional factors. Other DNA viruses, including phages, encode a single subunit RNA pol that is closer to DNA polymerases and reverse transcriptase than to multisubunit RNA pols [5]
RNA dependent RNA polymerases (RDRP)
Most RNA viruses that lack DNA stage in their cycle encode RNA-dependent RNA polymerases, the enzymes responsible for RNA replication through RNA duplex formation. This enzyme also has a single subunit and is structurally similar to other single subunit polymerases [6].
Most RNA viruses that lack DNA stage in their cycle encode RNA-dependent RNA polymerases, the enzymes responsible for RNA replication through RNA duplex formation. This enzyme also has a single subunit and is structurally similar to other single subunit polymerases [6].
PolyA Polymerases (PAP)
Polyadenosine polymerase or polynucleotide adenylyltransferase [7] may formally be classified as RNA polymerase, since it also builds polyribonucleotide chain, adding poly(A) tail to the 3'-end of pre mRNA.
Table. The comparison of different RNA polymerase types
RNA Polymerase in Transcription: stages
- Initiation: The DNA templates for transcription contain specific regulatory sequences called promoters. Gene-specific and some universal transcription factors recognize these sequences and help RNA pol binding to template and to change its conformation from "closed" to "open". In open complex DNA double-stranded molecule is partially unwound and forms a transcription bubble.
- Elongation: During this phase, the RNA strand lengthens within the DNA/RNA heteroduplex. As rNTPs are incorporated into the chain, the template DNA strand unwinds downstream in 5'–3' direction, while the upstream DNA strand "rewinds" to its initial state, releasing the newly formed RNA from the duplex. RNA pol employes Mg2+-dependent mechanism on this step, as DNA polymerases do.
- Termination: The final step of transcription may depend on rho-factor that destabilizes elongation complex, or may be regulated by "stop" palindrome sequence within the template DNA.
RNA polymerase in biotechnology
In biotechnology the most widespread RNA pols applied are the bacteriophages T7 and SP6 enzymes. These polymerases have been well-studied and are convenient for in vitro transcription and gene engineering because of their high fidelity and relatively compact size (compared to bacterial enzyme or RNA polymerase II eukaryotes). T7 RNA polymerase does not require additional factors to initiate in vitro transcription and is stimulated by spermidine. To produce RNA molecules, DNA vectors are created with a phage promoter sequence added before the desired gene. T7 polymerase are mainly applied to synthesize mRNA and guide RNA for gene editing systems.
The commercially available recombinant T7RNA polymerases possess a high processivity thanks to gene selection, and could effectively incorporate labeled and modified rNTPs. Additionally ready-to-use transcriptional kits with well-combined reagents (e. g. T7 High-yield RNA synthesis kit, Biolabmix) have been gaining popularity.
The commercially available recombinant T7RNA polymerases possess a high processivity thanks to gene selection, and could effectively incorporate labeled and modified rNTPs. Additionally ready-to-use transcriptional kits with well-combined reagents (e. g. T7 High-yield RNA synthesis kit, Biolabmix) have been gaining popularity.