RNA Polymerase

1.0What is RNA polymerase?

RNA polymerase (RNAP), also known as DNA-directed RNA polymerase (DdRP), is an enzyme responsible for synthesizing RNA from a DNA template. With the assistance of helicase, RNAP locally unwinds double-stranded DNA, allowing one strand to serve as a template for RNA synthesis, known as transcription. Before initiation, a transcription factor and its associated mediator complex must bind to a promoter region. RNAP not only initiates transcription but also guides nucleotides, aids in attachment and elongation, possesses proofreading and replacement capabilities, and recognizes termination signals.

RNAP synthesizes two types of RNA: messenger RNA (mRNA) for protein coding and non-coding RNA genes, including transfer RNA (tRNA), ribosomal RNA (rRNA), and microRNA (miRNA).

2.0Structure of RNA Polymerase

RNA polymerases catalyzing transcription are intricate, multimeric proteins. The RNA polymerase of E. coli consists of two alpha (𝛂) subunits, each weighing 36 kDa, a beta (𝛃) subunit of 150 kDa, a beta prime subunit (𝛃') of 155 kDa, with a small omega (𝛚) factor. The complete RNA polymerase holoenzyme, 𝛂𝛂𝛃𝛃'𝛚𝛔, includes sigma. The sigma subunit guides the enzyme to transcription initiation sites, after which it dissociates, leaving the core enzyme (𝛂𝛂𝛃𝛃'𝛚) to catalyze elongation. The alpha (𝛂) and beta (𝛃) subunits contribute to the tetrameric core's assembly, while the beta' (𝛃') subunit binds the DNA template. All RNA polymerases (RNAPs) contain essential metal cofactors, notably zinc and magnesium cations, crucial for facilitating the transcription process.

3.0Function of RNA Polymerase

The primary function of RNA polymerase is to catalyze the synthesis of RNA molecules from DNA templates through a process called transcription in both prokaryotes and eukaryotes. RNA polymerase (RNAP) initiates transcription at specific DNA sequences called promoters. It synthesizes an RNA chain complementary to the template DNA strand through elongation. In eukaryotes, RNAP can generate RNA chains as long as 2.4 million nucleotides, such as the full length of the dystrophin gene. At the end of genes, RNAP preferentially releases its RNA transcript at specific DNA sequences known as terminators.

4.0RNA polymerase Products

RNA polymerase produces various RNA molecules with diverse functions:

• Messenger RNA (mRNA): Acts as a template for protein synthesis by ribosomes.
• Non-coding RNA or "RNA genes": Encode RNA not translated into protein, including tRNA and rRNA involved in translation. Recent discoveries suggest RNA genes play broader roles.
• Transfer RNA (tRNA): Transfers specific amino acids to growing polypeptide chains during protein synthesis at ribosomes.
• Ribosomal RNA (rRNA): Constituent of ribosomes, essential for protein synthesis.
• MicroRNA: Regulates gene activity.
• Catalytic RNA (Ribozyme): Enzymatically active RNA molecules.

5.0Components of RNA polymerase

In bacteria, RNA polymerase (RNAP) synthesizes both mRNA and non-coding RNA (ncRNA). RNAP is a large enzyme consisting of five subunits. The subunits include 𝛃, 𝛃' two 𝛂 subunits, and 𝛚, the smallest subunit. These subunits play crucial roles in RNA synthesis, promoter interaction, and enzyme assembly and stabilization.

The RNA polymerase (RNAP) core unit combines with the transcription initiation factor sigma (σ) to create the RNA polymerase holoenzyme 𝛂𝛂𝛃𝛃'𝛚σ. Sigma enhances the holoenzyme's ability to recognize promoters accurately, reducing its affinity for nonspecific DNA while increasing specificity for promoter regions. Consequently, transcription begins at the appropriate sites. 

RNA Polymerase in Prokaryotes

• In Prokaryotes, only one type of RNAP is involved in the synthesis of different types of RNA (rRNA, mRNA, tRNA, etc). RNAP, also referred to as RNA Polymerase Holoenzyme, is a multicomplex enzyme with a molecular weight of 465kD. The polymerisation rate of RNAP in the majority of the prokaryotes is 40-50 nucleotides per second.
• Being a multicomplex enzyme, it’s made up of ve different subunits (polypeptides). 
• The RNAP holoenzyme is divided into two components, namely the core enzyme and the sigma factor. 
• The core enzyme includes two same α, β, β’ and ω subunits. 
• The sigma factor includes σ subunits. RNAP holoenzyme can be inhibited by an antibiotic called RIFAMPICIN.

Now let us look at all the subunits individually on what roles they play and what are the genes responsible for encoding them.

α subunit –

• It is encoded by a gene called rpoA gene. 
• They are in pairs i.e., two alpha subunits. It is located in the core enzyme.
• Its function is to bind to the promoter region and is involved in regulation of transcription. Molecular weight is about 41Kd.

β subunit –

• They are encoded by rpo B genes. Molecular weight is 155kd. Located in the core enzyme.
• Functions include a catalytic site and binds to NTPs (nucleotide tri-phosphates), such as ATP, UTP, GTP etc. Inhibited by Rifampicin, an Antibiotic which inhibits the holoenzyme not the core enzyme.

β’ subunit –

• They are encoded by rpo C gene and have molecular weight of 165 Kd. Located in the core enzyme. 
• It facilitates the catalytic activity after binding to the DNA template strand. Generally, it binds to Zn ions.

ω subunit – 

• Encoded by rpo Z gene. Located in the core enzyme. Functions include assembly of RNA polymerase.
• It is the smallest subunit as its molecular weight is about 10kd. It contains 91 Amino acids.

σ subunit –

• This subunit is encoded by rpo D genes (sigma 70). located in the holoenzyme. 
• Functions include, recognition of promoters and increase the Affinity of holoenzyme to bind to promoters. + The σ is known as the “housekeeping” factor, because it initiates transcription of most genes.
• Bacterial cells have different types of σ factors which are encoded by different types of genes and also different functions.
• σ is encoded by rpoN gene and involved in nitrogen regulated gene transfer. 
• σ is encoded by rpoS gene and involves in gene expression during the stationary and starvation phase. 
• σ is encoded by rpoH gene and is involved in heat shock gene transcription.

RNA Polymerase in Eukaryotes 

In eukaryotes, there are five different types of RNA polymerase from which three are main, they are RNA polymerase I, RNA polymerase II and RNA polymerase III.

The identification of these RNA polymerases was done using a toxin known as α-amanitin. α-amanitin is extracted i.e. isolated from a poisonous mushroom called Amanita phalloids. It is made up of eight amino acids and is a cyclic peptide. It is also known as Death cap.

• RNA polymerase II and III are inhibited by this toxin, whereas RNA polymerase I is not.
•  RNA polymerase II is highly sensitive and is inhibited at concentration of 1μg/ml of αamanitin. 70 54 38 32.
•  RNA polymerase III is moderately sensitive and is inhibited at concentration of 10μg/ml of α-amanitin.
•  All the eukaryotic RNA polymerases have molecular mass more than 500kDa.

Eukaryotes possess multiple types of nuclear RNA polymerases (RNAP), each dedicated to synthesizing specific subsets of RNA. These include:

RNA Polymerase In Archaea 

RNA polymerase in archaea shares similarities with both eukaryotic RNA polymerase II and bacterial RNA polymerase. It is responsible for transcribing various types of RNA, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and other non-coding RNAs. The structure and function of archaeal RNA polymerase exhibit unique features compared to its counterparts in bacteria and eukaryotes, reflecting the distinct evolutionary lineage of archaea.