RNA

While DNA polymerases are the DNA builders, RNA Polymerases are the crucial enzymes that transcribe genetic information from a DNA template into an RNA molecule. They are the engines driving gene expression and are vital reagents in the molecular lab for synthesizing RNA in vitro

Think of them as specialized molecular photocopiers that read a DNA blueprint and produce an RNA working copy

Core Function: DNA-Dependent RNA Synthesis

The fundamental reaction catalyzed by RNA polymerases is the synthesis of an RNA strand complementary to a DNA template strand

1. Template Binding The polymerase recognizes and binds to a specific DNA sequence called a promoter, located upstream of the gene/sequence to be transcribed
2. DNA Unwinding The polymerase locally unwinds the DNA double helix near the promoter, creating a “transcription bubble” and exposing the template strand
3. Initiation RNA polymerase begins synthesizing the RNA chain using ribonucleoside triphosphates (NTPs: ATP, UTP, CTP, GTP) as substrates. Crucially, unlike DNA polymerases, RNA polymerases DO NOT require a primer to start synthesis. They can initiate de novo
4. Elongation The polymerase moves along the DNA template strand (reading it 3’ → 5’), adding complementary NTPs to the 3’-hydroxyl end of the growing RNA strand (synthesizing 5’ → 3’). U pairs with A in the DNA template
5. Termination Synthesis continues until the polymerase encounters a specific terminator sequence in the DNA, signaling it to release the newly synthesized RNA transcript and dissociate from the DNA template

Essential Requirements for RNA Polymerase Activity (In Vitro)

When using RNA polymerase as a reagent in the lab, you need:

• DNA Template: Must contain the sequence to be transcribed downstream of a promoter sequence recognized by the specific RNA polymerase being used. Can be linear (e.g., PCR product, restriction fragment) or circular (plasmid)
• RNA Polymerase Enzyme: The specific polymerase enzyme (e.g., T7, T3, SP6 RNA polymerase)
• Ribonucleoside Triphosphates (NTPs): ATP, UTP, CTP, and GTP as building blocks and energy source. Labeled NTPs (e.g., biotin-UTP, DIG-UTP, ³²P-UTP) can be included for probe synthesis
• Divalent Cations: Usually Magnesium ions (Mg²⁺) as an essential cofactor
• Appropriate Buffer: To maintain optimal pH, salt concentration, and potentially include agents like DTT (reducing agent) or RNase inhibitors

Types of RNA Polymerases Used as Reagents

While cells have complex RNA polymerases (e.g., Pol I, II, III in eukaryotes; multi-subunit enzyme in bacteria), the most commonly used RNA polymerases as reagents in the molecular lab are derived from bacteriophages due to their simplicity and high specificity:

• Phage RNA Polymerases (T7, T3, SP6)
  • Source: Bacteriophages T7, T3, and SP6, respectively. These viruses infect bacteria
  • Structure: Relatively simple, typically single-subunit enzymes (compared to complex cellular polymerases)
  • Promoter Specificity: This is their key advantage! Each phage polymerase recognizes its own unique and highly specific promoter sequence (e.g., T7 RNA polymerase only initiates transcription efficiently from a T7 promoter). This allows for precise control over which sequence is transcribed from a DNA template engineered to contain that specific promoter
  • Efficiency: They are generally very active and processive, capable of synthesizing large amounts of RNA from the template
  • Usage: These are the workhorses for In Vitro Transcription (IVT) reactions. Plasmids used for IVT often contain multiple cloning sites flanked by different phage promoters (e.g., T7 on one side, SP6 on the other), allowing transcription of the inserted gene in either direction (sense or antisense) depending on which polymerase is used
• Bacterial RNA Polymerase (e.g., E. coli)
  • Structure: More complex multi-subunit enzyme (core enzyme + sigma factor for promoter recognition)
  • Promoter Specificity: Recognizes bacterial promoters (e.g., -10 and -35 consensus sequences). Requires the sigma factor for specific initiation
  • Usage: Less commonly used for routine IVT compared to phage polymerases due to its complexity and less defined promoter requirements in simple in vitro systems. However, it’s fundamentally important for understanding transcription and is used in specialized research applications
• Eukaryotic RNA Polymerases (Pol I, II, III)
  • Structure: Very complex, multi-subunit enzymes requiring numerous additional protein factors (General Transcription Factors - GTFs) for promoter recognition and initiation
  • Usage: Almost never used as simple biochemical reagents for routine IVT in clinical or standard molecular labs due to their complexity and dependence on multiple GTFs. Their study is critical, but they aren’t practical tools for simply making RNA in a test tube without reconstituting a complex system

Key Properties for Lab Applications

When selecting or using an RNA polymerase reagent:

• Promoter Specificity: Ensures transcription starts where intended
• Yield/Efficiency: Important for generating sufficient amounts of RNA probe, standard, or therapeutic mRNA
• Processivity: Ability to synthesize long transcripts without premature termination
• Fidelity: While generally lacking proofreading, the accuracy of transcription is still a factor
• Ability to Incorporate Modified NTPs: Important for labeling or creating modified RNAs

Clinical Laboratory Relevance & Applications

Phage RNA polymerases are essential reagents for various applications:

• In Vitro Transcription (IVT): The primary use. Generating RNA molecules in a test tube
  • Probe Synthesis: Creating labeled RNA probes (e.g., with biotin, digoxigenin, fluorescent dyes, or historically radioactivity) for hybridization techniques:
    • Northern Blotting: Detecting specific RNAs in a sample
    • In Situ Hybridization (ISH/FISH): Locating specific RNAs within cells or tissues
    • Microarrays: Used in some protocols for sample labeling
    • (Antisense probes are often generated for higher specificity)
  • Synthesis of RNA Standards and Controls: Creating known amounts of specific RNA transcripts to use as positive controls or for absolute quantification in RT-qPCR assays (e.g., quantifying viral load or gene expression)
  • mRNA Synthesis for Research & Therapeutics
    • Producing mRNA for in vitro translation experiments
    • Generating functional mRNAs for studies on RNA processing, localization, or function
    • Crucially, large-scale IVT using phage polymerases (especially T7) is the basis for producing mRNA vaccines (like Pfizer/BioNTech and Moderna COVID-19 vaccines) and emerging mRNA therapeutics.: (This often involves co-transcriptional addition of a 5’ cap analog and enzymatic addition of a poly(A) tail post-transcription)
  • Synthesis of Functional RNAs: Producing tRNAs, rRNAs, ribozymes, or regulatory RNAs (like guide RNAs for CRISPR systems) for research
• Coupled Transcription-Translation Systems: Some commercial kits provide phage RNA polymerase mixed with cell-free extracts (containing ribosomes, tRNAs, etc.) allowing direct protein synthesis from a DNA template carrying the appropriate phage promoter

Distinction from DNA Polymerase

It’s vital to remember the key differences:

Feature	DNA Polymerase	RNA Polymerase
Template	DNA	DNA
Product	DNA	RNA
Substrates	dNTPs (dATP, dGTP, dCTP, dTTP)	NTPs (ATP, GTP, CTP, UTP)
Primer Req.	Yes (needs free 3’-OH to extend)	No (can initiate de novo)
Proofreading	Often Yes (3’→5’ exonuclease)	Generally No
Main Use	Replication, PCR, Sequencing	Transcription, IVT (Probe/RNA synth)

Key Terms

• RNA Polymerase: Enzyme synthesizing RNA from a DNA template
• Transcription: The process of synthesizing RNA from DNA
• Promoter: DNA sequence recognized by RNA polymerase to initiate transcription
• NTPs (Ribonucleoside Triphosphates): Building blocks of RNA (ATP, UTP, CTP, GTP)
• In Vitro Transcription (IVT): Synthesis of RNA in a test tube using purified components
• Phage RNA Polymerase (T7, T3, SP6): Simple, efficient RNA polymerases recognizing specific phage promoters; workhorses for IVT
• Antisense Probe: An RNA probe complementary to the target mRNA sequence
• mRNA Vaccine: Vaccine using IVT-synthesized mRNA encoding a viral antigen
• Template Strand: The DNA strand read by the RNA polymerase (3’→5’)
• Coding Strand: The DNA strand whose sequence resembles the RNA transcript (with T instead of U)