RNA polymerases (RNAP or RNApol) are found in all organisms and many viruses. Bacteria have a single RNA polymerase while there are three RNA polymerases involved in eukaryotic cells. This functional protein is made up of 5 subunits i.e. α2, β, β1, ω and σ. Where α2 β β1 and ω are the core polymerase subunits and sigma-factors assists the polymerase to recognize the promoter. In prokaryotes, all the RNA molecules are transcribed by a single RNA polymerase. RNAP opens the double-stranded DNA so that its one strand of nucleotides can be used as a template for the synthesis of RNA, a process called transcription
Core Enzyme The α-subunits are required for assembly of core enzyme. It may have role in recognition of promoter specially the upstream element of few gene. The β and β1 subunits together form the catalytic center.
The functional sites of core enzyme have
• Binding site for sense strand of DNA.
• Binding site for antisense strand of DNA.
• Site for unwinding of DNA.
• DNA rewinding site.
• RNA binding site.
σ-Factor It ensures the stable binding of RNA polymerase. Holoenzyme only at promoters and thus it ensures promoter recognition generally it recognizes -35 and -10 regions.
•It increases the binding specificity of RNA polymerase.
• It is involved only in initiation; once transcription is initiated it gets released.
• It increases affinity of the core enzyme to the promoter.
Generally in prokaryotes three consensus sequences are present which are described below
• -35 Sequence It is recognized by σ factor having consensus sequence TTGACA.
• -10 Sequence It is the unwinding domain of promoter as it is rich in AT base pairs and thus requires minimum energy to melts and also called pribnow box.
• Start Point The point from which transcription starts, mostly a purine of a triplet CAT.
Transcription in bacteria is carried out by RNA polymerase, which must bind to the sigma factor to initiate transcription.
• The sigma factor associates with the core enzyme to form a holoenzyme, which binds to the –35 and –10 consensus sequences in the promoter, creating a closed complex.
• The holoenzyme binds the promoter tightly and unwinds the double-stranded DNA, creating an open complex.
• A nucleoside triphosphate complementary to the DNA at the start site serves as the first in the RNA molecule.
• Two phosphates are cleaved from each subsequent nucleoside triphosphate, creating an RNA nucleotide that is added to the 3‘end of the growing RNA molecule.
• The sigma factor is released as the RNA polymerase moves beyond the promoter.
Eukaryotic RNA polymerases
Eukaryotes have multiple types of RNA polymerases in their nuclei-RNA polymerase I, II III, IV and V. Each nuclear RNA polymerase is a large protein with about 8-14 subunits and molecular weight approximately 50,000 for each. Each polymerase has different functions. Polymerases are responsible for transcribing most genes. Pol I and Pol III are involved in transcribing specialized RNA encoding genes.
RNA polymerase I specifically transcribes the large ribosomal RNA precursor gene whereas
RNA polymerase II synthesizes precursors of mRNAs and most snRNA and microRNAs.
RNA polymerase III synthesizes transfer RNAs, ribosomalRNA 5S and other small RNAs found in the nucleus and cytosol.
RNA polymerase IV synthesizes siRNA in plants.
RNA polymerase V synthesizes RNAs involved in siRNA-directed heterochromatin formation in plants.
Steps involved during transcription by RNA polymerase In order to transcribe a gene, RNA polymerase proceeds through three phases as described below:
• Initiation A promoter is the DNA sequence that initially binds the RNA polymerase. The promoter-polymerase complex undergoes structural changes required for initiation to proceed. Like DNA replication, transcription proceeds in a 5ˈ-3ˈ direction. A ribonucleotide is added to the 3ˈ end of the growing chain. As RNA polymerase binds promoters in a defined orientation, the same strand is always transcribed from a given promoter. The choice of promoter determines which stretch of DNA is to be transcribed and is the key step at which helix regulation is imposed. Steps involved in initiation of transcription
i. Closed complex Once after the initial binding of polymerase to the promoter, DNA remains double stranded and the enzyme binds to one face of the helix.
ii. Open complex The closed complex undergoes a transition to the open complex in which the DNA strands separate over a distance of around 14bp around the start site to form a transcription bubble. The template strand is freed as the DNA opens up. The first two ribonucleotides are brought into the active site, aligned and joined together in the template strand.
iii. Stable ternary complex This complex is formed when DNA, RNA and the enzyme is about to proceed towards elongation.
• Elongation After synthesis of short stretch of RNA, it shifts into elongation phase. The transition gives the polymerase a grip the template more firmly. The enzyme in this phase unwinds the DNA in front and reanneals it from behind, it dissociates the growing RNA chain from the template and it performs proofreading as well.
• Termination The release of RNA product occurs in this phase.