Transcription and Posttranscriptional Processes: Study Notes

Transcription and Posttranscriptional Processes

Introduction

This chapter covers the molecular mechanisms by which genetic information is transferred from DNA to RNA and subsequently processed in cells. It explores the enzymology of transcription, regulation of gene expression, and the posttranscriptional modifications that occur in both prokaryotic and eukaryotic systems.

Information Transfer in the Cell

Central Dogma and Key Processes

• DNA Replication: The process by which DNA is duplicated prior to cell division, ensuring genetic continuity. It is template-directed and highly accurate.

• Transcription: The synthesis of RNA from a DNA template. This process is catalyzed by RNA polymerase and is the first step in gene expression.

• Reverse Transcription: The synthesis of DNA from an RNA template, as seen in retroviruses.

• Translation: The process by which mRNA is used as a template to synthesize proteins.

• Processivity vs. Fidelity: Processivity refers to how far a polymerase can synthesize a nucleic acid chain without dissociating, while fidelity refers to the accuracy of nucleotide incorporation.

Regulation of Transcription by DNA-Protein Interactions

Gene Expression Control

• Gene expression is regulated by proteins that recognize specific DNA sequences (regulatory elements) and bind to them.

• These DNA-binding proteins can act as activators (enhancing transcription) or repressors (inhibiting transcription) of downstream genes.

• Regulation is essential for cellular differentiation and response to environmental signals.

The Operon Model

Jacob and Monod's Operon Model (1961)

• The operon is a cluster of genes under the control of a single promoter and operator, allowing coordinated regulation.

• Key components:

  • Regulator gene: Encodes a repressor protein.

  • Operator: DNA sequence where the repressor binds.

  • Structural genes: Encode proteins with related functions.

  • Inducer: Small molecule that can inactivate the repressor, allowing gene expression.

• Example: The lac operon in E. coli regulates lactose metabolism.

Experiments Elucidating RNA

RNA Characterization Techniques

• Density Gradient Centrifugation: Separates nucleic acids based on density, allowing distinction between DNA and RNA.

• Sucrose Gradient Centrifugation: Separates molecules based on size and shape, useful for analyzing ribosomal RNA and other RNA species.

• RNA is denser than DNA due to its higher base composition and structural differences.

Enzymology of RNA Synthesis: RNA Polymerase

RNA Polymerases in Prokaryotes and Eukaryotes

• Prokaryotes: Have a single RNA polymerase composed of multiple subunits (α, β, β', σ, ω).

• Eukaryotes: Possess three distinct RNA polymerases:

  • RNA polymerase I: Synthesizes rRNA.

  • RNA polymerase II: Synthesizes mRNA and some small RNAs.

  • RNA polymerase III: Synthesizes tRNA and other small RNAs.

• Each polymerase requires specific transcription factors for initiation.

Subunit Composition of E. coli RNA Polymerase

Subunit	Molecular Weight (kDa)	Number per Enzyme	Function
α	36.5	2	Chain initiation, interaction with regulatory proteins
β	151.0	1	Chain initiation and elongation
β'	155.0	1	DNA binding
σ	Varies	1	Promoter recognition
ω	10.0	1	Enzyme assembly

Mechanism of Transcription in Bacteria

Initiation

• RNA polymerase binds to the promoter region, forming a closed complex.

• DNA unwinds at the -10 region, forming an open complex.

• Initiation requires Mg2+ and the correct NTPs.

Elongation

• RNA chain grows by addition of ribonucleotides complementary to the DNA template.

• Phosphodiester bonds are formed by nucleophilic attack of the 3'-OH group of the growing RNA on the α-phosphate of the incoming NTP.

• The transcription bubble moves along the DNA as synthesis proceeds.

$\text{(RNA)_{n}} + \text{NTP} \rightarrow \text{(RNA)_{n+1}} + \text{PP}_i$

Termination

• Factor-independent termination: Involves formation of a GC-rich hairpin loop in the RNA, followed by a series of U residues, causing dissociation.

• Factor-dependent termination: Requires proteins such as Rho (ρ) to release the RNA transcript from the polymerase.

Transcription in Eukaryotic Cells

Key Differences from Prokaryotes

• Greater complexity and regulation due to chromatin structure (nucleosomes).

• Genes are often interrupted by introns and are not organized in operons.

• Transcription and translation are compartmentalized (nucleus vs. cytoplasm).

Eukaryotic Promoters and Transcription Factors

• Promoters contain conserved elements such as the TATA box (analogous to the -10 region in prokaryotes).

• Enhancer regions can be located far from the transcription start site and increase transcription efficiency via DNA looping.

• Transcription factors (TFI, TFII, TFIII) are required for initiation by RNA polymerases I, II, and III, respectively.

Chromatin Modification and Transcriptional Activity

• Acetylation of histone lysine residues reduces their positive charge, weakening DNA-histone interactions and promoting transcription.

• High levels of histone acetylation are associated with active gene expression.

Termination in Eukaryotes

• RNA polymerase II transcribes beyond the end of the gene, passing through polyadenylation signals (e.g., AAUAAA).

• The pre-mRNA is cleaved downstream of the signal, and a poly(A) tail is added by poly(A) polymerase.

• Poly(A) tails enhance mRNA stability and translation efficiency.

Posttranscriptional Processing

5' Capping

• Eukaryotic pre-mRNA is capped at the 5' end with 7-methylguanosine.

• This cap protects mRNA from degradation and is involved in ribosome binding during translation.

Splicing

• Introns are removed from pre-mRNA by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs).

• Splicing joins exons to produce mature mRNA.

• Alternative splicing allows a single gene to produce multiple protein isoforms.

Example: Alternative Splicing of the α-Tropomyosin Gene

• The α-tropomyosin gene in rats undergoes alternative splicing to generate different mRNAs in various tissues.

• This increases protein diversity without increasing gene number.

Tools of Biochemistry: Chromatin Immunoprecipitation (ChIP)

Genome-Wide Identification of DNA-Binding Proteins

• ChIP is a technique used to identify the binding sites of DNA-associated proteins across the genome.

• It involves crosslinking proteins to DNA, immunoprecipitating the complexes, and sequencing the associated DNA.

• This method is essential for studying transcription factor binding and chromatin structure.