Transcription in prokaryotes and eukaryotes

Content
 Definition and significance in gene expression.
 DNA template and RNA synthesis (5'→3').
 RNA polymerase, initiation, elongation, termination, and operons in
prokaryotes.
 RNA polymerases (I, II, III), transcription factors, and post-
transcriptional modifications (capping, splicing, polyadenylation) in
eukaryotes.
 Prokaryotic operons and eukaryotic enhancers/silencers in
transcription regulation.
 Role of rRNA, tRNA, miRNA, and other non-coding RNAs in gene
expression.
 Introduction to Transcription
 What is a Gene?
o A gene is a sequence of DNA that contains the instructions to make proteins or RNA
molecules. These instructions are carried out via transcription (RNA synthesis) and
translation (protein synthesis).
o Genes are responsible for the biological functions of cells and organisms.
 Types of RNA:
o mRNA (Messenger RNA): Carries genetic information from DNA to ribosomes for
protein synthesis.
o rRNA (Ribosomal RNA): A component of ribosomes where protein synthesis occurs.
o tRNA (Transfer RNA): Transfers amino acids to the ribosome, matching them to the
mRNA code.
 o miRNA (Micro RNA): Regulates gene expression by inhibiting translation or promoting
degradation of specific mRNAs.
o snRNA (Small nuclear RNA): Involved in RNA splicing to produce mature mRNA.
o Other non-coding RNAs: Includes snoRNA and lncRNA, which regulate gene
expression and participate in RNA processing.
 Gene Regulation:
Gene expression is tightly controlled at the level of transcription. Promoters, enhancers, and
silencers are key sequences that regulate gene expression.
Transcription Process Overview
 What is Transcription?
o Transcription is the synthesis of RNA from a DNA template. This occurs in the nucleus
in eukaryotes and in the cytoplasm in prokaryotes.
o RNA polymerase reads the DNA in the 3′ to 5′ direction, synthesizing RNA in the 5′ to
3′ direction, where RNA replaces thymine (T) with uracil (U).
Transcription Mechanism
 Template DNA Strand:
o RNA polymerase reads the antisense strand (template strand) to synthesize a
complementary RNA molecule. The coding strand (sense strand) has the same
sequence as the RNA (except T is replaced with U).
 Polymerization Process:
o RNA polymerase adds nucleotides to the 3′ end of the growing RNA molecule,
forming phosphodiester bonds. The reaction is powered by the hydrolysis of high-
energy phosphate bonds from RNA nucleotides (rNTPs), releasing pyrophosphate
(PPi).
RNA Polymerase Structure
 Bacterial RNA Polymerase:
o Composed of a core enzyme (α₂, β, β′, ω) and a sigma factor (σ) that helps recognize
promoter sequences.
o Once RNA synthesis begins, the sigma factor is released, and the core enzyme
continues elongation.
 Eukaryotic RNA Polymerases:
o Eukaryotes have three RNA polymerases:
1. RNA polymerase I: Transcribes rRNA (except 5S).
2. RNA polymerase II: Transcribes mRNA and most snRNA.
3. RNA polymerase III: Transcribes tRNA and 5S rRNA.
o RNA polymerase II requires transcription factors to initiate transcription at the
promoter.
Figure: Bacterial RNA polymerase, during elongation, unwinds downstream DNA, synthesizes
complementary RNA, and guides the nascent RNA (red) out through a channel in the β
subunit. The β′, β, and α subunits coordinate DNA and RNA processing, while the ω subunit
stabilizes the structure.
 Prokaryotic vs. Eukaryotic Transcription
Prokaryotic Transcription:
 Operons: Genes in prokaryotes are often arranged in operons, transcribed
together into a single mRNA.
 Coupled Transcription and Translation: Transcription and translation occur
simultaneously in the cytoplasm.
 Limited RNA Processing: No extensive post-transcriptional processing;
mRNA is typically ready for translation.
Eukaryotic Transcription:
 Gene Structure: Eukaryotic genes are split into exons (coding regions) and
introns (non-coding regions), which need splicing.
 RNA Processing: Eukaryotic mRNA undergoes capping, splicing, and
polyadenylation before it can be translated.
 Transcription and Translation Separation: Transcription happens in the
nucleus; translation occurs in the cytoplasm.
 Stages of Transcription
TRANSCRIPTION IN PROKARYOTES
Transcription in prokaryotes occurs in the cytoplasm and involves three stages: initiation,
elongation, and termination. Each step is mediated by specific factors and mechanisms.
INITIATION
1.Promoter Recognition:
I. RNA polymerase holoenzyme binds to the promoter region.
II. The prokaryotic promoter consists of two conserved sequences:
a) -10 region (Pribnow box): TATAAT sequence.
b) -35 region: TTGACA sequence.
1. Sigma (σ) factor: Recognizes the promoter sequence and helps RNA polymerase
bind to it.
2.Closed Complex Formation:
RNA polymerase, along with the σ-factor, binds to the double-stranded DNA at the
promoter region, forming a closed complex.
3.Open Complex Formation:
RNA polymerase unwinds about 12-14 base pairs of DNA near the -10 region, forming
the transcription bubble.
4.Initial Transcription (Abortive Initiation):
RNA polymerase synthesizes short RNA transcripts (~10 nucleotides). These may
dissociate initially until the polymerase escapes the promoter.
5.Promoter Escape:
The σ-factor is released after initiation, allowing RNA polymerase to transition to
elongation mode.
Figure: In RNA transcription, the start site is designated as +1, with downstream bases numbered
positively and upstream bases negatively. The template DNA strand guides RNA synthesis, producing
RNA complementary to it and identical to the non-template strand (except U replaces T).
• Formation of the First Phosphodiester Bond:
 The RNA polymerase synthesizes the first nucleotide of RNA. The first ribonucleotide is added
to the 3′ end of the RNA strand, forming a phosphodiester bond between the first two
ribonucleotides.
 In prokaryotes, the transcription complex initiates with a short RNA primer synthesized by
the polymerase.
 In eukaryotes, additional regulatory proteins may help stabilize the complex before the
enzyme begins full elongation.
Figure: RNA polymerase catalyzes
RNA synthesis in the 5′→3′
direction by forming
phosphodiester bonds between
the 3′ end of the growing RNA
strand and complementary rNTPs,
guided by template DNA base
pairing.
ELONGATION
1.RNA Synthesis:
a) Core RNA polymerase (without σ-factor) moves along the template strand in the 3'
to 5' direction, synthesizing RNA in the 5' to 3' direction.
b) The nucleotide triphosphates (NTPs) are added complementary to the DNA
template.
2.Transcription Bubble Maintenance:
RNA polymerase maintains a transcription bubble of about 15 nucleotides and a short
RNA-DNA hybrid (~8 nucleotides).
3.Proofreading Mechanisms:
a) Pyrophosphorolytic editing: Removal of incorrect nucleotides using pyrophosphate.
b) Hydrolytic editing: RNA polymerase backtracks, cleaves the error-containing RNA,
and resumes transcription.
• Proofreading and Error Correction:
 RNA polymerase can proofread the RNA it is synthesizing, using a
backtracking mechanism to correct errors by removing incorrect
nucleotides.
 However, RNA polymerase has a lower fidelity than DNA polymerase and
does not always correct every error, resulting in some RNA mutations.
TERMINATION
o Termination is the final stage of transcription, where RNA polymerase stops synthesizing
RNA, dissociates from the DNA, and releases the newly formed RNA transcript.
o The process of termination differs between prokaryotes and eukaryotes.
• There are two mechanisms of termination in prokaryotes:
1.Rho-Independent (Intrinsic) Termination:
a) Involves a GC-rich palindromic sequence followed by a poly-U sequence in the RNA.
b) The RNA folds into a hairpin structure, destabilizing the RNA-DNA hybrid and causing RNA
polymerase to dissociate.
2.Rho-Dependent Termination:
a) The Rho protein, an ATP-dependent helicase, binds to the rut (Rho utilization) site on RNA.
b) Rho moves along the RNA, catches up with RNA polymerase at the termination site, and
unwinds the RNA-DNA hybrid, leading to termination.
TRANSCRIPTION IN EUKARYOTES
Transcription in eukaryotes occurs in the nucleus and involves RNA polymerase I, II, or III, depending on the type
of RNA transcribed. Each step (initiation, elongation, termination) is more complex than in prokaryotes and
involves multiple factors.
INITIATION
1.Chromatin Remodeling:
a) Histone acetyltransferases (HATs) acetylate histones, loosening chromatin structure.
b) Chromatin remodeling complexes reposition nucleosomes to expose promoter regions.
2.Promoter Recognition:
Eukaryotic promoters have:
a) Core promoter elements: TATA box, Initiator (Inr), and Downstream Promoter Element (DPE).
b) Regulatory sequences: Enhancers and silencers.
3.Pre-Initiation Complex (PIC) Formation:
General Transcription Factors (GTFs):
a) TFIID: Recognizes and binds the TATA box via its TATA-binding protein (TBP) subunit.
b) TFIIB, TFIIE, TFIIF, TFIIH: Assemble sequentially, stabilizing RNA polymerase II binding.
c) Mediator Complex: Acts as a bridge between activators and RNA polymerase II.
4.Open Complex Formation:
a) TFIIH (helicase activity): Unwinds DNA, forming the transcription bubble.
b) TFIIH (kinase activity): Phosphorylates the C-terminal domain (CTD) of RNA polymerase II, activating it
for elongation.
5.Promoter Escape:
Phosphorylation of CTD causes RNA polymerase II to release most GTFs and begin RNA synthesis.
ELONGATION
1.RNA Synthesis:
RNA polymerase II synthesizes RNA in the 5' to 3' direction, using NTPs.
2.Elongation Factors:
a) DSIF and NELF: Pause RNA polymerase II briefly for quality control.
b) P-TEFb: Resumes elongation by phosphorylating DSIF, NELF, and the CTD of RNA
polymerase II.
3.Co-Transcriptional Processing:
a) Capping: Addition of a 5' cap (7-methylguanosine).
b) Splicing: Removal of introns by the spliceosome.
c) Polyadenylation: Addition of a poly-A tail at the 3' end.
TERMINATION:
 RNA polymerase II (which transcribes mRNA) terminates transcription after the RNA is cleaved
at a specific site, and a poly-A tail is added to the 3′ end of the transcript.
 The cleavage and polyadenylation specificity factor (CPSF) recognizes the polyadenylation
signal sequence (AAUAAA) in the RNA and cleaves the pre-mRNA. The remaining part of the
polymerase complex dissociates.
 Polymerase II then continues synthesizing RNA beyond the cleavage site until it eventually
dissociates.
• Release of RNA and Polymerase:
 In both prokaryotes and eukaryotes, once termination occurs, the RNA molecule is released, and
the RNA polymerase enzyme dissociates from the DNA template. The newly synthesized RNA
transcript is now free to undergo further processing (in eukaryotes) or translation (in both
prokaryotes and eukaryotes).
• Post-Transcriptional Modifications in Eukaryotes
 5′ Cap Addition:
o A 7-methylguanosine cap is added to the 5′ end of the nascent RNA
molecule to protect it from exonucleases, promote splicing, and assist
in translation initiation.
 RNA Splicing:
o The spliceosome, composed of snRNA and proteins, removes non-
coding introns from the pre-mRNA, leaving only the coding exons to
form the mature mRNA.
 Polyadenylation:
A poly-A tail is added to the 3′ end of the RNA, which enhances RNA stability
and facilitates nuclear export.