Introduction of RNA polymerase:
RNA polymerase, also known as DNA-directed/dependent RNA polymerase (DdRP), is usually known as RNAP, this enzyme creates RNA from a DNA template or RNApol in molecular biology.
Locally, the double-stranded DNA is opened by the enzyme RNAP using helicase so that one strand of the exposed nucleotides can serve as a template for the production of RNA, a procedure known as transcription. Before RNAP can start the DNA unwinding at a specific location, A DNA binding site known as a transcription mediator complex must be connected to a transcription factor.
Along with starting RNA transcription, RNAP also directs nucleotides into place, promotes attachment and elongation, has inherent proofreading and replacement capacities, and has the ability to recognize terminations. In eukaryotes, RNAP can build chains up to 2.4 million nucleotides long.RNAP creates RNA that is functionally either messenger RNA (mRNA), which codes for proteins, or non-coding (so-called “RNA genes”). There are four distinct kinds of functional RNA genes:
Transfer RNA (tRNA) functions as an enzymatically active RNA molecule, transferring particular amino acids to expanding polypeptide chains at the ribosomal site of protein synthesis during translation. Ribosomes include ribosomal RNA (rRNA). Gene activity is regulated by microRNAs (miRNA).
RNA polymerase is essential for life and is found in a wide variety of living things of viruses. An RNA polymerase can be a protein complex (multi-subunit RNAP) or just have one subunit (single-subunit RNAP, ssRNA), each of which represents a distinct lineage, depending on the organism. Due to their shared fundamental structure and action, the former can be found in bacteria, archaea, and eukaryotes.
The latter is found in phages and has a connection to modern DNA polymerases eukaryotic chloroplasts and mitochondria. Eukaryotic and archaeal RNAPs are controlled differently and have more subunits than bacterial ones. There is only one RNA polymerase in bacteria and archaea.
RNA polymerase in eukaryotes
Eukaryotes have various nuclear RNAP types, each of which is in charge of synthesizing a particular subset of RNA:
RNA polymerase 1
Pre-rRNA 45S (or 35S in yeast) is created by RNA polymerase I, and as it develops, it becomes the primary RNA section of the ribosome.
RNA polymerase 2
The majority of sRNA, microRNA, and mRNA precursors are produced by RNA polymerase II.
RNA polymerase 3
TRNAs, rRNA 5S, and other short RNAs that are present in the cytosol and nucleus are created by RNA polymerase III.
RNA polymerase 4&5
Less is known about RNA polymerase IV and V, which are found in plants and produce siRNA. The chloroplasts also encode and employ an RNAP that resembles bacteria in addition to the ssRNAPs.
What is the Structure of RNA Polymerase?
American biochemist Roger D. Kornberg showed a precise molecular representation of the RNAP enzyme at distinct transcriptional phases. Because of the demonstration, he received the Nobel Prize.
Two alpha subunits (36 kDa each), one beta subunit (150 kDa), one beta prime subunit (155 kDa), and a small-sized omega subunit make up the core of the only kind of RNA polymerase enzyme found in prokaryotes. To create a holoenzyme, the core enzyme joins forces with a sigma factor.
Along the length of the DNA that needs to be transcribed, the core enzyme creates a crab claw. Metal cofactors like zinc and magnesium are present in the RNA Pol enzymes and aid in transcription. The fundamental structure of eukaryotic RNAPs is identical to that of the enzyme, but they additionally contain a few more subunits.
Explain the function of RNA polymerase.
The core tenet of molecular biology has traditionally viewed RNA as a messenger molecule that exports the data encoded in DNA out of the nucleus to trigger the creation of proteins in the cytoplasm. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are two other prominent RNAs that are closely related to the machinery that produces proteins.
However, it has become increasingly obvious over the past 20 years that RNA has a variety of tasks, of which protein-coding is only one. Some even play a critical role in the development of gametes. Some regulate gene expression. They are known as non-coding RNAs or ncRNAs.
Regulating the quantity and type of RNA transcripts produced in response to the needs of the cell is one of RNAP’s primary jobs because it contributes to the synthesis of molecules with such a diverse spectrum of roles. The enzyme, particularly the carboxy-terminal end of one subunit, interacts with a variety of other proteins, transcription factors, and signaling molecules to control its activity.
This regulation is thought to have been essential for the emergence of eukaryotic plants and animals, which exhibit variable gene expression and cellular specialization despite having genetically similar cells. Additionally, effective transcription is necessary for these RNA molecules to perform at their best; the DNA template strand’s sequence must be faithfully translated into RNA.
In other areas, even a single base alteration can result in a wholly unusable product. As a result, even while the enzyme must operate swiftly and finish the polymerization reaction in a brief amount of time, it also needs reliable mechanisms to guarantee incredibly low mistake rates. The complementarity of the nucleotide substrate to the template DNA strand is checked at various stages.
The environment is favorable for catalysis and the elongation of the RNA strand when the proper nucleotide is present. Additionally, a proofreading stage enables the removal of inaccurate bases.RNA polymerases also play a role in the post-transcriptional alteration of RNAs to make them functional, which facilitates their export from the nucleus to their final location of activity.
Explain the process of RNA polymerase.
In bacteria, the core promoter region containing the 35 and 10 elements (placed before the beginning of the sequence to be transcribed) is recognized by the sigma factor, and at some promoters, the subunit C-terminal domain also recognizes promoter upstream elements. Sigma factors are interchangeable and each one recognizes a different set of promoters.
For instance, in E. coli, gene 70 is produced normally and can detect the promoters of genes that are needed in a typical environment (“housekeeping genes”), whereas 32 detects the promoters of genes needed in hot environments (“heat-shock genes”).
The actions of the bacterial general transcription factor sigma are shared by a number of cooperative general transcription factors in archaea and eukaryotes. The “transcription preinitiation complex” is the common name for the RNA polymerase-promoter closed complex. The RNA polymerase changes from a closed complex to an open complex after binding to the DNA.
The “transcription bubble” is an unwinding segment of DNA with a length of around 13 bp that results from this separation of the DNA strands. Due to the DNA’s unwinding and rewinding, supercoiling is crucial to polymerase activity. There are compensating positive supercoils because the DNA in the region in front of RNAP is not wound. Negative supercoils are present and regions behind RNAP are rewound.
Once ribonucleotides have been base-paired to the template DNA strand in accordance with Watson-Crick base-pairing interactions, RNA polymerase begins to create the initial DNA-RNA heteroduplex. RNA polymerase interacts with the promoter region, as was already mentioned. However, these stabilizing connections prevent the enzyme from accessing DNA later on and preventing the synthesis of the entire product.
RNA polymerase needs to get out of the promoter in order to continue the synthesis of RNA. While unwinding more downstream DNA for synthesis and “squeezing” more downstream DNA into the initiation complex, it must retain promoter connections. The term “stressed intermediate” refers to RNA polymerase during the promoter escape transition.
The actions that result in DNA unwinding and compaction create stress on a thermodynamic level. The RNA polymerase releases its upstream connections and successfully completes the promoter escape transition into the elongation phase once the DNA-RNA heteroduplex is long enough (10 bp). The elongation complex is kept stable by the heteroduplex at the active center.
The stress can also be reduced by RNA polymerase by releasing its downstream connections, which stops transcription. The halted transcribing complex has two options: either (1) release the nascent transcript and start over at the promoter, or (2) employ RNA polymerase’s catalytic activity to reestablish a new 3′-OH on the nascent transcript at the active site and restart DNA scrunching to achieve promoter escape.
Short RNA fragments of about 9 bp are produced as a result of abortive transcription, which is the unproductive cycling of RNA polymerase before the promoter escape transition. The intensity of the promoter contacts and the availability of transcription factors determine how much initiation is aborted.
The 8-bp DNA-RNA hybrid, or the 8 base pairs in which the RNA transcript is attached to the DNA template strand, is part of the 17-bp transcriptional complex. Ribonucleotides are added to the 3′ ends of the RNA transcript as transcription proceeds, and the RNAP complex travels along the DNA. Prokaryotes and eukaryotes typically elongate at rates between 10 and 100 cents per second.
The RNAP’s aspartyl (asp) residues will cling to Mg2+ ions, which will then coordinate the ribonucleotide phosphates. The incoming NTP’s -phosphate will be retained by the initial Mg2+. This enables the 3′-OH from the RNA transcript to be attacked nucleophilically, adding another NTP to the chain. The pyrophosphate of the NTP will be held onto by the second Mg2+. The general reaction formula is:
NMPn+1 + PPi→ (NMP)n + NTP
Rho-dependent or rho-independent RNA transcription termination is a possibility in bacteria. The former depends on the rho factor, which triggers RNA release by destabilizing the DNA-RNA heteroduplex. The latter often referred to as intrinsic termination, is supported by a palindromic DNA region. When the area is transcribed, the RNA transcription loops and binds to itself, forming a “hairpin” structure. Because it frequently contains several G-C base pairs, this hairpin structure is more stable than the DNA-RNA hybrid itself.
The transcription complex’s 8 bp DNA-RNA hybrid changes into a 4 bp hybrid as a result. The entire RNA transcript will separate from the DNA because the final 4 base pairs are weak A-U base pairs. Though less well understood than in bacteria, eukaryotic transcription termination requires cleaving the new transcript and adding adenines to its new 3′ ends through a process known as polyadenylation.
Explain the components of RNA Polymerase.
The RNAP enzymes in bacteria create both mRNA and non-coding RNA. Five subunits make up the big molecule that is the enzyme:
β’:It is the biggest component, made up of part of the active center that produces RNA.
β:It is the second-largest subunit and is made up of the remaining active center material.
ɑ:There are two copies of the third-largest subunit, which are: ɑI and ɑII. It is the smallest subunit which makes it easier for the RNA Pol enzyme to be put together.
⍵: To create the RNAP holoenzyme, the RNA Pol interacts with the transcription initiation factor. As a result, RNAP’s affinity for non-specific DNA binding is decreased while its affinity for binding to promoters is increased.
All of the RNAs in Archaea are produced by a single type of RNAP. Its structural resemblance to RNA Pol II, a component of bacterial and eukaryotic RNAP, is striking.
Comparison between RNA and DNA:
Although both DNA and RNA polymerases accelerate nucleotide polymerization processes, there are two key differences between the two in terms of how active they are. RNAP enzymes, unlike DNA polymerases, do not require a primer to start the polymerization reaction.
Additionally, they have the ability to start the reaction in the midst of a DNA strand and read “STOP” signals, which leads the enzyme complex to separate from the template. Finally, RNA polymerases have the advantage of only needing to make a complimentary copy of one strand of DNA, albeit being a little slower than their counterparts.