RNA Transcription in Prokaryotes & Eukaryotes

RNA Transcription in Prokaryotes & Eukaryotes

RNA Structure and Types

• RNA vs. DNA - Recap

  • RNA: Ribonucleic acid; DNA: Deoxyribonucleic acid

  • RNA uses Uracil (U) instead of Thymine (T)

  • Physical differences due to a single oxygen atom

• DNA vs. RNA – II

  • Both use phosphodiester linkages

  • RNA is mostly single-stranded, unlike double-stranded DNA

  • RNA structures have different geometries

• DNA vs. RNA - III

  • RNA major groove is deeper and narrower than DNA

  • RNA double helix core is open and less stable than DNA double helix

  • Lower stability drives RNA secondary structure formation

• RNA Secondary Structure

  • Forms complex secondary structures by base-pairing with itself.

  • Conventional and non-conventional base pairs (e.g., A-C/G, C-A/U)

  • RNA structure & function are interconnected

• Diversity of Cellular RNAs

  • mRNA (messenger RNA)

  • tRNA (transfer RNA)

  • rRNA (ribosomal RNA)

  • lncRNA (long non-coding RNA)

  • miRNA (micro RNA)

Central Dogma and Transcription Overview

• Central Dogma of Molecular Biology

  • Transcription (Weeks 13 + 14)

  • Translation (Weeks 15 + 16)

  • Replication (Weeks 10 + 11)

  • RNA decodes DNA sequence into amino acid sequence (protein)

• Transcription - I

  • Like DNA replication, RNA synthesis occurs in the 5' to 3' direction.

  • Only one DNA strand is copied each time

  • Gene → Transcription → RNA → Translation → Protein

• Transcription - II

  • Template strand is copied 3’ to 5’

  • RNA does not base-pair with DNA after synthesis

  • New nucleotides added to the 3’ -OH group of the previous nucleotide

• Direction of Transcription

  • Only one strand acts as the DNA template at any given time.

  • Cell decides where to start and which direction to go

Stages of Transcription

• Three Stages of Transcription

  • Initiation: RNA polymerase binds to duplex DNA and unwinds it at the promoter.

  • Elongation: Polymerase synthesizes RNA.

  • Termination: RNA polymerase and RNA are released.

Prokaryotic Transcription

• Prokaryotic RNA Polymerase

  • Core RNA polymerase: α + β + β’ + ω factors

  • RNA polymerase holoenzyme: Core RNA polymerase + σ factor

  • Each subunit has a specific role.

  • One RNA polymerase in prokaryotes.

  • Different sigma factors add different functionality.

• Prokaryotic Transcription Initiation

  • Sigma factor helps in template recognition.

  • Closed complex forms at the promoter.

  • DNA unwinding leads to an open complex.

  • Polymerase “scrunches” the DNA during initiation.

  • Abortive initiation: stalls at 3-8 nucleotides.

  • Productive initiation: proceeds beyond 10 nucleotides.

  • Contest between sigma-promoter interaction and DNA tension

• Transcription Elongation

  • RNA polymerase moves along the template strand, unwinding and rewinding DNA.

  • Nascent RNA is synthesized using ribonucleoside triphosphates.

  • Prokaryotic transcription occurs at 50 nucleotides/second.

  • 12 nt window of RNA-DNA hybrid

• Multiple Initiations

  • Multiple RNA polymerases can transcribe the same gene simultaneously.

• Transcription Termination

  • Prokaryotic mRNAs can be polycistronic or monocistronic

  • Two main mechanisms:

    • Rho(ρ)-independent: most common

    • Rho(ρ)-dependent: half of “factor-dependent” terminators

• Rho-Independent Termination

  • Requires a G-C-rich region in stem and a single-stranded U-run.

  • Hairpin in RNA may be required, leading to RNA polymerase and RNA release.

  • Almost all sequences required for termination are in the transcribed region.

• Rho-Dependent Termination

  • Rho monomer has two domains: N-terminal RNA-binding domain and C-terminal ATPase domain.

  • Rho attaches to the rut site on RNA and translocates along it.

  • RNA polymerase pauses at hairpin, and rho catches up.

  • Rho unwinds DNA-RNA hybrid, leading to termination.

• Transcriptional Regulation

  • Prokaryotes control the level of transcript by using different promoter sequences.

• Promoter Consensus Sequences

  • A consensus sequence is the “average” sequence found when comparing many sequences.

• Alternative Promoters

  • Different sigma factors recognize different promoter sets.

  • Substitution of sigma factor causes enzyme to recognize different promoters.

  • Examples: Standard promoter, Heat-shock promoter, Nitrogen-starvation promoter

  • Different situations require different gene regulations.

  • Examples of Gene Factors: rpoD (σ^{70}), rpoS (σ^{S}), rpoH (σ^{32}), rpoE (σ^{E}), rpoN (σ^{54}), rpoF (σ^{FecI})

Eukaryotic Transcription

• Eukaryotic Transcription: Introduction

  • In eukaryotes, transcription and translation are separated by the nucleus.

• Eukaryotic RNA Polymerases

  • RNA polymerase I: transcribes 5.8S, 18S, and 28S rRNA genes

  • RNA polymerase II: transcribes all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, and most snRNA genes

  • RNA polymerase III: transcribes tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs

• Eukaryotic RNA Polymerase Structure

  • Some components are the same for eukaryotic and prokaryotic polymerases, while others are divergent.

• Transcription Initiation - Eukaryotes

  • TATA box at -30 in humans

  • TFII = Transcription Factor for polymerase II

  • TBP = TATA binding protein

• Transcription Initiation – Eukaryotes Continued

  • Initiation complex: RNA pol II, TFIIA, B, D, E, F, H

  • TFIIH phosphorylates CTD

  • TFIIA, B, D remain on promoter

  • As in prokaryotes – abortive + productive initiation

• Eukaryotic Elongation & Termination

  • Elongation factors provide speed and processivity.

  • Mechanism similar to prokaryotes.

  • Termination requires polyA tailing – discussed next week.

• General Transcription Factors

  • Needed for all genes

  • Eukaryotes also use specialized transcription factors

• Eukaryotic vs Prokaryotic Transcription

  • Eukaryotes:

    • Transcription in the nucleus, translation in the cytoplasm

    • RNA processing (capping, splicing, polyadenylation)

    • Uncoupled transcription/translation

  • Bacteria:

    • Confined to cytosol

    • No RNA processing

    • Coupled transcription/translation

Summary

• RNA can adopt various structures linked to its function.

• Initiation involves promoter sequence recognition by RNA polymerase.

• Elongation involves nucleotide addition.

• Termination in prokaryotes requires a hairpin and can be factor-dependent or independent.