Prokaryotic and Eukaryotic Transcription: Mechanisms and Regulation

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Prokaryotic Transcription

Overview of Prokaryotic Transcription

Transcription in prokaryotes is the process by which RNA is synthesized from a DNA template. This process is essential for gene expression and is tightly regulated to ensure cellular efficiency and adaptability.

Key Enzyme: RNA polymerase (RNAP) is responsible for synthesizing RNA.
Stages: Transcription consists of three main stages: initiation, elongation, and termination.

Sigma Factor Switching

Sigma factors are specialized proteins that associate with the core RNA polymerase to form the holoenzyme, directing it to specific promoter sequences.

Competition: Multiple sigma factors compete for the core enzyme.
Expression Level: The predominant sigma factor used depends on its expression level in the cell.
Environmental Response: For example, σ70 is dominant during normal growth, but under heat shock, σ32 becomes prevalent, leading to the expression of heat-shock genes.

Sigma Factor	-35 Sequence	Separation	-10 Sequence
σ70	TTGACA	16-18 bp	TATAAT
σ54	GGGAAAT	11-12 bp	GCTGATAA
σ32	CCCTTGAA	13-15 bp	CCGGAINT
σ38	CTGGNA	15-17 bp	TTGGA
σ28	AGGANP	11-12 bp	GCCGATAA
Additional info:	Other sigma factors exist for specific stress responses.

Stages of Prokaryotic Transcription

Transcription in prokaryotes proceeds through three distinct stages:

Initiation: Assembly of the transcription apparatus at the promoter. This is often the rate-limiting step and subject to regulation.
Elongation: RNA polymerase synthesizes the RNA strand by adding nucleotides.
Termination: RNA polymerase and the newly synthesized RNA are released from the DNA template.

Prokaryotic Initiation

Initiation involves the formation of a closed complex, where the RNA polymerase holoenzyme binds to the promoter region without unwinding the DNA.

Closed Complex: Duplex DNA interacts with the sigma factor, typically from -55 to +1 relative to the transcription start site.
Reversibility: RNAP binding to the promoter is reversible at this stage.
Transition to Open Complex: The DNA unwinds at the -10 region (TATA box), allowing the template strand to enter the active site of the enzyme.

Example: The sigma factor recognizes specific promoter sequences, facilitating the recruitment of RNAP to the correct transcription start site.

Abortive Initiation and Promoter Escape

During early transcription, RNA polymerase may synthesize and release short RNA fragments (abortive initiation) before successfully escaping the promoter to begin elongation.

Abortive Initiation: Short RNA chains are repeatedly synthesized and released.
Promoter Escape: Once RNAP successfully clears the promoter, elongation proceeds.
Rate-Limiting Step: Promoter escape is often the rate-limiting step in transcription initiation.

Eukaryotic Transcription

Overview of Eukaryotic Transcription

Eukaryotic transcription is more complex than prokaryotic transcription, involving multiple RNA polymerases and extensive regulation by transcription factors and chromatin structure.

RNA Polymerases: Eukaryotes have three main RNA polymerases: Pol I, Pol II, and Pol III.
Chromatin Structure: DNA is packaged into nucleosomes, affecting accessibility for transcription.

RNA Polymerases in Eukaryotes

Polymerase	Transcribes
Pol I	rRNA (18S, 5.8S, 28S)
Pol II	mRNA, some small RNAs (microRNA)
Pol III	tRNA, 5S rRNA, other small RNAs (snRNA)

Promoter Structure and Transcription Initiation

Eukaryotic promoters are defined as binding sites for RNA polymerase and transcription factors. The core promoter is essential for basal transcription, while upstream elements and enhancers modulate expression levels.

Core Promoter: Contains elements such as the TATA box, initiator (Inr), and downstream promoter elements (DPE).
Upstream Elements: CAAT box, GC box, and other motifs that bind specific transcription factors.
Enhancers and Silencers: Regulatory DNA sequences that increase or decrease transcription, often located far from the promoter.

General Transcription Factors (GTFs) and Preinitiation Complex (PIC)

Transcription initiation by Pol II requires the assembly of a preinitiation complex (PIC) composed of general transcription factors and RNA polymerase II.

Key GTFs: TBP (TATA-binding protein), TFIIB, TFIIE, TFIIF, TFIIH.
TFIIH: Has kinase and helicase activities; phosphorylates the C-terminal domain (CTD) of Pol II and unwinds DNA.
CTD of Pol II: Contains repeats of the sequence YSPTSPS, which are phosphorylated during transcription initiation and elongation.

Example: The TATA box is recognized by TBP, which helps recruit other GTFs and Pol II to the promoter.

Termination and RNA Processing

Unlike prokaryotes, eukaryotic transcription termination does not rely on a specific terminator sequence. Instead, a polyadenylation signal (AAUAAA) directs cleavage and polyadenylation of the pre-mRNA.

Polyadenylation: Addition of a poly(A) tail (80-250 nucleotides) to the 3' end of mRNA, enhancing stability and export.
RNA Processing: Includes capping, splicing, and polyadenylation, all coupled with transcription.

Comparison: Prokaryotic vs. Eukaryotic Transcription

Feature	Prokaryotes	Eukaryotes
Promoter Elements	-10 (Pribnow box), -35	TATA box, Inr, DPE, CAAT, GC box
RNA Polymerases	Single RNAP	Pol I, II, III
Initiation Factors	Sigma factors	General transcription factors (GTFs)
Chromatin Structure	Absent	Present (nucleosomes)
Termination	Terminator sequence	Polyadenylation signal

Key Terms

Holoenzyme: The complete RNA polymerase complex, including the sigma factor.
Promoter: DNA sequence where transcription machinery assembles.
Sigma Factor: Protein that directs RNAP to specific promoters in prokaryotes.
General Transcription Factors: Proteins required for transcription initiation in eukaryotes.
CTD (C-terminal domain): Repetitive region of Pol II, subject to phosphorylation.

Formulas and Equations

Promoter Recognition:
CTD Repeat Sequence: where is the number of repeats (e.g., 52 in humans)

Example Application

During heat shock, E. coli switches from using σ70 to σ32, enabling the transcription of genes that help the cell survive elevated temperatures.

Additional info: These notes expand on the brief points in the original slides, providing definitions, context, and examples for key concepts in transcription relevant to Genetics students.