RNA Polymerase Definition

What is RNA Polymerase?

RNA polymerase (RNApol or RNAP) is an enzyme that uses a single-stranded DNA or RNA template to build RNA by linking ribonucleoside triphosphates (NTPs) together with phosphodiester bonds. It's also called transcriptase because it copies genetic information from genes into RNA. It needs four NTPs (ATP, GTP, CTP, UTP) and metal ions like Mg²⁺ or Mn²⁺ to work. The reaction it catalyzes is: (NMP)_n + NTP → (NMP)_n+1 + PPi. RNA synthesis happens from 5' to 3', starting with a nucleotide with three phosphate groups. As each nucleotide is added, a pyrophosphate group is released, forming a bond, and the release of pyrophosphate drives the process. Unlike DNA polymerase, RNA polymerase doesn't need a primer and can start RNA synthesis directly. It can locally unwind DNA, simplifying transcription, but it can't proofread.

T7 RNA Polymerase

T7 RNA polymerase, weighing about 99 kDa, catalyzes RNA formation from 5' to 3'. It's highly specific to T7 promoters and can't recognize others. Using double-stranded DNA with the T7 promoter sequence and NTPs, it synthesizes RNA complementary to the downstream single-stranded DNA, making it useful for in vitro synthesis of long and short transcripts. Double-stranded linear blunt-ended or 5' overhanging end DNA can serve as templates for T7 RNA polymerase, allowing linear plasmids and PCR products to be used as templates for in vitro RNA synthesis.

Illustration of the two different conformational states presented by T7 RNAP during the catalytic process. (Dousis, A.; et al, 2023)

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RNA Polymerase Structure

RNA polymerases in prokaryotes and eukaryotes share common characteristics, but they also differ in structure, composition, and properties.

Prokaryotic RNA Polymerase

The prokaryotic RNA polymerase best studied is from Escherichia coli, comprised of six subunits (α2ββ'ωσ) with a total molecular weight of approximately 500,000. The core enzyme, α2ββ'ω, combines with the σ factor to form the holoenzyme. The σ factor's main role is to recognize the DNA promoter region, facilitating holoenzyme binding. It initiates transcription by partially unwinding the DNA double helix upon binding to specific promoter sequences. Seven σ factors exist in E. coli, with σ70 primarily recognizing housekeeping gene promoters. Environmental changes induce specific σ factor production, initiating gene transcription. Rifampicin and rifamycin are antibiotics that selectively inhibit prokaryotic RNA polymerases by binding to the β subunit. Even if added after transcription begins, they hinder transcription, indicating the β subunit's role throughout the process. Similar RNA polymerase structures and functions are found in other prokaryotes.

Eukaryotic RNA Polymerase

Eukaryotic RNA polymerases are structurally more intricate than their prokaryotic counterparts. They comprise two large subunits, two α-like subunits, and one ω-like subunit, analogous to the β and β' subunits, and two α subunits, and the ω subunit found in Escherichia coli RNA polymerase. Additionally, each of the three eukaryotic RNA polymerases contains 7 to 11 small subunits. Unlike prokaryotic cells, which can carry out transcription solely with RNA polymerase subunits, eukaryotic cells rely on additional protein factors and processing modifications for transcription. Eukaryotic mitochondria also possess their own RNA polymerase responsible for synthesizing mitochondrial mRNA, tRNA, and rRNA. The activity of mitochondrial RNA polymerase can be inhibited by rifampicin or rifamycin, akin to prokaryotic RNA polymerase.

Types of RNA Polymerase

Initially, researchers classified eukaryotic RNA polymerases into three types based on their sensitivity to α-amanitin, namely RNA polymerase i, ii, and iii. Among them, RNA polymerase I shows insensitivity to α-amanitin, RNA polymerase II exhibits sensitivity to low doses of α-amanitin, while RNA polymerase III displays sensitivity to high doses of α-amanitin. Consequently, α-amanitin has long been regarded as a lethal toxin. Upon entering the human body, it is promptly taken up by liver cells, where it rapidly binds to RNA polymerase II and RNA polymerase III, leading to the dissolution of liver cells and ultimately resulting in permanent liver damage.

RNA Polymerase i

RNA polymerase i primarily synthesizes the precursor of 45S ribosomal RNA (pre-rRNA), which later matures into 28S, 18S, and 5.8S ribosomal RNA. These ribosomal RNAs are essential components of ribosomes, crucial for protein synthesis within cells.

RNA Polymerase ii

RNA polymerase II is primarily responsible for synthesizing precursor messenger RNA (mRNA), along with most small nuclear RNA (snRNA) and microRNA. Its activity requires the cooperation of various transcription factors, which bind to the promoter region of genes, facilitating the initiation of transcription.

RNA Polymerase iii

RNA polymerase III is mainly involved in synthesizing transfer RNA (tRNA), 5S ribosomal RNA (rRNA), and some other small nuclear and cytoplasmic RNAs. These molecules play essential roles in protein synthesis and other cellular processes.

In plants, subsequent discoveries led to the identification of RNA polymerase IV and RNA polymerase V. RNA polymerase IV is responsible for synthesizing small interfering RNAs (siRNAs), which play regulatory roles in gene expression. On the other hand, RNA polymerase V is involved in generating RNAs associated with the formation of heterochromatin, directed by siRNAs, thereby contributing to genome stability and regulation in plants.

What is the Role of RNA Polymerase in Transcription?

RNA polymerase is a vital enzyme in transcription, converting DNA templates into RNA molecules. This process is fundamental to organism function and is primarily facilitated by RNA polymerases. Here's an overview of their key functions:

Promoter Recognition

RNA polymerase identifies specific DNA sequences, called promoters, to initiate transcription. Once bound to the promoter, transcription commences.

RNA Strand Synthesis

Bound to the promoter, RNA polymerase synthesizes the RNA strand by integrating nucleotides sequentially from the DNA template, a process known as elongation. Transcription ceases upon encountering a terminator sequence, releasing the synthesized RNA product.

Selective Nucleotide Initiation

RNA polymerase selectively uses ATP, GTP, UTP, and CTP as initiating nucleotides for transcription. Their interactions enable the chemical reactions needed to initiate RNA synthesis.

DNA Deconjugation

Unlike DNA polymerases, RNA polymerases possess deconjugating enzyme activity, eliminating the need for additional enzymes to unwind the DNA double helix during transcription.

RNA polymerases play crucial roles in promoter recognition, RNA strand synthesis, transcription termination, and selective nucleotide initiation. Their activity is regulated by factors such as promoter structure, transcription factors, and other regulatory mechanisms.

DNA Polymerase vs RNA Polymerase

DNA polymerases and RNA polymerases are vital enzymes involved in genetic processes within cells. DNA polymerases, comprising types I, II, III, IV, and V, primarily elongate DNA strands during replication. On the other hand, RNA polymerase synthesizes RNA using DNA as a template, sharing key catalytic features with DNA polymerase:

• Both use DNA as a template.
• Both catalyze nucleic acid synthesis via polymerization.
• Both form phosphodiester bonds between nucleotides.
• Both read the template 3' to 5' and synthesize RNA 5' to 3'.
• Both faithfully transcribe according to base pairing.

While, here are the differences between DNA polymerase and RNA polymerase:

• Functions: RNA polymerase transcribes RNA, while DNA polymerase replicates DNA.
• Primer Requirements: RNA polymerase doesn't need primers, unlike DNA polymerase.
• Polymerase Activity: RNA polymerase works in the 5' to 3' direction only, while DNA polymerase also has 3' to 5' exonuclease activity, enhancing replication fidelity.
• Deconvolution Capability: RNA polymerase untwists DNA, whereas DNA polymerase requires assistance from other enzymes during replication.
• Substrate Specificity: RNA polymerase uses ribonucleoside triphosphate (NTP), while DNA polymerase uses deoxyribonucleoside triphosphate (dNTP).

Applications of RNA Polymerase

RNA polymerase is a pivotal enzyme in the process of gene expression. Its primary role lies in transcribing DNA into RNA, a crucial step in the central dogma of molecular biology. This process involves the unwinding of the DNA double helix and the synthesis of a complementary RNA strand based on the sequence of the template DNA.

• The applications of RNA polymerase are diverse and profound across various fields of study and industry. In basic research, it serves as a fundamental tool for studying gene regulation, transcriptional dynamics, and RNA processing. Researchers utilize RNA polymerase to synthesize RNA molecules for various purposes, such as generating probes for hybridization studies, creating RNA standards for quantitative assays, and producing RNA transcripts for functional studies in vitro and in vivo.
• In biotechnology and medicine, RNA polymerase finds applications in gene therapy, where it is used to produce therapeutic RNA molecules, such as mRNA vaccines or RNA interference (RNAi) therapeutics, for treating diseases. Additionally, RNA polymerase is integral in the field of synthetic biology, enabling the engineering of synthetic gene circuits and the construction of artificial genetic systems.
• In industrial settings, RNA polymerase plays a crucial role in bioproduction processes. It is employed in the synthesis of RNA molecules for various applications, including the production of recombinant proteins, enzymes, and pharmaceuticals. Moreover, RNA polymerase-based systems are utilized in the development of biosensors and diagnostic assays for detecting nucleic acids and monitoring gene expression levels.

Overall, the versatility and utility of RNA polymerase make it an indispensable tool in molecular biology, biotechnology, medicine, and various other scientific disciplines, driving advancements in research, healthcare, and industrial bioprocessing.

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Reference

1. Dousis, A.; et al. An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Nature Biotechnology. 2023, 41: 560 - 568.

RNA Polymerase Definition

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