HMG - Week 10: Transcription and Gene Expression

Gene Expression and the Central Dogma

Definition of Gene Expression: This is the biological process through which information encoded within a DNA sequence is utilized to create a functional product, which is typically a protein.
The Two Major Steps of Gene Expression:
- Transcription: The process of converting DNA into RNA ( $DNA \rightarrow RNA$ ).
- Translation: The process of converting RNA into a protein ( $RNA \rightarrow Protein$ ).
The Central Dogma:
- Proposed by Francis Crick in $1956$ , this concept describes the unidirectional flow of genetic information: $DNA \rightarrow RNA \rightarrow Protein$ .
Importance of Gene Expression:
- Enables cells to produce necessary proteins.
- Allows cells to respond dynamically to environmental changes.
- Facilitates cell specialization (differentiation) into distinct cell types.
- Regulates essential processes such as metabolism and growth.
- Key Principle: Although almost all cells in an organism contain the identical DNA genome, the specific genes expressed vary significantly between different cell types.

The Transcription Process

Definition: Transcription is the synthesis of an RNA molecule using a specific DNA sequence as a template.
Primary Enzyme: RNA Polymerase is the enzyme responsible for this process. Its primary functions include:
- Binding to the DNA molecule.
- Opening the DNA double helix (unwinding the strands).
- Synthesizing the RNA strand.
- Note: Only one of the two DNA strands acts as the template during any single transcription event.

Types of RNA Molecules Produced

mRNA (Messenger RNA):
- Serves as the carrier of genetic information from the DNA in the nucleus to the ribosomes in the cytoplasm.
- Acts as the direct template for protein synthesis during translation.
tRNA (Transfer RNA):
- Responsible for carrying specific amino acids to the ribosome during translation.
- Contains anticodons that pair with mRNA codons to ensure the correct amino acid sequence.
rRNA (Ribosomal RNA):
- Provides both structural and catalytic (enzymatic) components to the ribosomes.
snRNA (Small Nuclear RNA):
- Vital for RNA processing and splicing.
- Forms a core part of the spliceosome.

The Three Stages of Transcription

These stages are conserved in both prokaryotic and eukaryotic organisms:

Initiation: RNA polymerase identifies and binds to the DNA at a specific site to begin the synthesis of RNA.
Elongation: RNA polymerase traverses the DNA template, adding complementary RNA nucleotides to the growing chain.
Termination: The process of RNA synthesis concludes, and the RNA polymerase enzyme detaches from the DNA template.

Transcription in Bacteria (Prokaryotes)

Model Organism: The lecture focuses on E. coli as the primary model for prokaryotic transcription.
Structure of a Prokaryotic Gene:
1. Promoter: The specific DNA sequence where RNA polymerase binds; it establishes the starting point for transcription.
2. RNA-Coding Region: The segment of DNA that is actually transcribed into an RNA sequence.
3. Terminator: The DNA sequence that signals the end of the transcription process.

Initiation in Prokaryotes

RNA Polymerase Holoenzyme: In bacteria, the functional enzyme complex consists of two main parts:
- Core Enzyme: The component that performs the actual synthesis of RNA.
- Sigma ($\sigma$) Factor: A regulatory protein responsible for recognizing specific promoter sequences and positioning the RNA polymerase correctly at the start site.
- Formula: $\text{Core Enzyme} + \sigma \text{ Factor} = \text{RNA Polymerase Holoenzyme}$ .
Promoter Recognition (Consensus Sequences):
- $-35$ Region: A consensus sequence located $35$ bases upstream of the start site with the sequence $5'\text{-TTGACA-}3'$ .
- $-10$ Region (Pribnow Box): A consensus sequence located approximately $10$ bases upstream of the transcription start site with the sequence $5'\text{-TATAAT-}3'$ .
The Two-Step Binding Process:
- Step 1: Closed Complex: The RNA polymerase holoenzyme binds loosely to the $-35$ region. At this stage, the DNA remains double-stranded.
- Step 2: Open Complex: The enzyme binds tightly to the $-10$ region, causing the unwinding of approximately $17$ base pairs. This creates the transcription bubble.
Positioning: The enzyme is now correctly aligned at the $+1$ transcription start site.
Promoter Clearance: Once approximately $8-9$ nucleotides have been successfully synthesized, the Sigma factor dissociates from the complex, and the core enzyme continues the elongation process independently.

Elongation and Termination

Mechanism of Elongation:
- RNA polymerase moves along the DNA, reading the template strand in the $3' \rightarrow 5'$ direction.
- The synthesis of RNA occurs in the $5' \rightarrow 3'$ direction.
- Base Pairing Rules: DNA $\rightarrow$ RNA
  - $A \rightarrow U$ (Uracil replaces Thymine in RNA)
  - $T \rightarrow A$
  - $G \rightarrow C$
  - $C \rightarrow G$
- Actions of RNA Polymerase during Elongation:
  1. Opens the DNA helix ahead of the synthesis point.
  2. Adds complementary RNA nucleotides.
  3. Reforms (rewinds) the DNA helix behind the moving enzyme.
  4. Releases the newly synthesized RNA strand as it is produced.
Termination:
- Occurs when the enzyme encounters a specific Terminator Sequence.
- Results: Transcription stops, the RNA transcript is released, and the RNA polymerase dissociates from the DNA.

Comparison: Replication vs. Transcription

Feature	Replication	Transcription
Template	Both DNA strands	Only one DNA strand
Region Copied	The entire genome	A single gene (or specific set of genes)
Primary Enzyme	DNA Polymerase	RNA Polymerase
Primer Required	Yes	No
Product	Double-stranded DNA ( $dsDNA$ )	Single-stranded RNA ( $ssRNA$ )
Bases Used	$A, T, G, C$	$A, U, G, C$
Proofreading	Extensive	Limited

Sigma Factors and Gene Regulation

Function: Sigma factors are proteins that enable RNA polymerase to recognize specific promoters. By utilizing different sigma factors, bacteria can direct RNA polymerase to specific sets of genes.
Regulatory Importance: This mechanism allows bacteria to adapt to environmental changes, such as stress, by expressing specific groups of genes simultaneously.
Sigma 70:
- The primary sigma factor in E. coli.
- Controls the transcription of "housekeeping" genes (genes required for basic cellular function).
- Most abundant and active during normal growth conditions.
Alternative Sigma Factors: Produced under specialized conditions, including:
- Heat shock.
- Starvation.
- General environmental stress.

Eukaryotic RNA Polymerases

Unlike prokaryotes which use a single RNA polymerase core, eukaryotes possess three distinct types:

RNA Polymerase I: Synthesizes most types of rRNA.
RNA Polymerase II: Synthesizes mRNA and certain snRNAs.
RNA Polymerase III: Synthesizes tRNA and the 5S rRNA.
Utility of Multiple Polymerases: This division of labor allows for greater control over gene expression, specialization of the transcription machinery, and increased cellular complexity.

Post-Transcriptional Modifications in Eukaryotes

1. 5' Modification (5' Capping)

Process: Shortly after transcription starts, a modified guanine nucleotide, known as the 7-methylguanosine cap (m7G cap), is added to the $5'$ end of the pre-mRNA.
Functions:
- Protection: Shields the transcript from degradation by exonucleases.
- Ribosome Recognition: Essential for the initiation of translation.
- Nuclear Export: Assists in the transport of the mRNA out of the nucleus.
- Splicing: Facilitates the efficient processing of the RNA.

2. 3' Modification (Polyadenylation)

Process: Following the cleavage of the pre-mRNA transcript, the enzyme Poly-A polymerase adds a string of adenine nucleotides to the $3'$ end, creating a Poly-A tail.
Scale: Typically consists of $50-250$ adenine nucleotides.
Functions:
- Stability: Increases the lifespan of the mRNA by protecting it from degradation.
- Nuclear Export: Facilitates transport to the cytoplasm.
- Translation Efficiency: Enhances the rate of protein production.

Pre-mRNA Processing and Splicing

Eukaryotic Gene Components:
- Exons: Coding sequences that will be expressed in the final protein.
- Introns: Non-coding intervening sequences that do not contribute to the final protein.
RNA Splicing: The biological process where introns are excised and exons are ligated together to produce mature mRNA suitable for translation.
The Spliceosome:
- A complex made of snRNAs and proteins known as snRNPs (Small Nuclear Ribonucleoproteins, pronounced "snurps").
- Roles: Recognizes intron-exon boundaries, removes introns, and joins exons together.
Importance of Splicing:
- Ensures the production of functional mRNA.
- Allows for Alternative Splicing: Different combinations of exons from a single gene can be joined together in various ways. This results in one gene encoding multiple distinct proteins, serving as a major contributor to protein diversity in humans.