SCH2226 Human Molecular Genetics - Gene Expression: Transcription

Topic = Transcription

The Central Dogma: Proposed by Francis Crick in 1956, this concept describes the flow of genetic information from: $\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}$ .
Definition of Transcription: The synthesis of an RNA molecule using a DNA template.
Key Characteristics:
Only one of the DNA strands is transcribed.
The process is catalyzed by the enzyme RNA polymerase.

Stages of Transcription: Transcription is divided into three distinct steps in both prokaryotes and eukaryotes:
1. Initiation: The process of getting started where RNA polymerase binds to the DNA.
2. Elongation: RNA polymerase adds nucleotides to the growing chain.
3. Termination: Transcription stops, and the RNA polymerase dissociates from the DNA.

Four Main Types of RNA:
Messenger RNA ( $\text{mRNA}$ ): Carries genetic information for protein synthesis.
Transfer RNA ( $\text{tRNA}$ ): Contains an anticodon and carries amino acids (attached via an ester bond to the $\text{ACC}$ sequence at the 3'-end).
Ribosomal RNA ( $\text{rRNA}$ ): Components of the ribosome.
Small Nuclear RNA ( $\text{snRNA}$ ): Essential for eukaryotic pre-mRNA processing.

Directionality: The RNA chain is synthesized in the $5' \rightarrow 3'$ direction.
Template: Only one strand of the DNA is used as a template to determine the base sequence.
Denaturation: The DNA double helix is opened/denatured by RNA polymerase in prokaryotes.
Initiation and the Sigma ( $\sigma$ ) Factor:
Initiation requires a complex consisting of RNA polymerase and the $\sigma$ factor protein binding to the promoter.
The $\sigma$ factor guides the RNA polymerase to recognize specific promoter sequences.
Once transcription begins (after 8 to 9 nucleotides are polymerized), the $\sigma$ factor dissociates from the core enzyme, and the core enzyme completes the synthesis.

Regions of a Prokaryotic Gene:
Promoter: A sequence that attracts RNA polymerase to begin transcription at a specific site.
RNA-coding sequence: The sequence that is actually transcribed.
Terminator: Specifies where transcription will stop.

Promoter Details and Consensus Sequences:
$-35$ Region: The consensus sequence is $5'\text{-TTGACA-}3'$ . The holoenzyme binds loosely here first.
$-10$ Region: Also known as the Pribnow box; the consensus sequence is $5'\text{-TATAAT-}3'$ RNA polymerase binds tightly here, causing a local untwisting of approximately $17\,bp$ .
$+1$ Site: The specific position where transcription initiation begins.

Gene Regulation:
Transcription levels depend on the ability of the $\sigma$ factor to recognize promoter sequences.
Promoters deviating from the consensus sequence show different transcription levels.
$\sigma^{70}$ : The most abundant $\sigma$ factor in $E. coli$ , used for most genes. Other factors are produced in response to changing environmental conditions.

Eukaryotic RNA Polymerases:
RNA Polymerase I: Located in the nucleolus; transcribes the three major rRNAs ( $28S$ , $18S$ , and $5.8S$ ).
RNA Polymerase II: Located in the nucleus; transcribes messenger RNA ( $\text{mRNA}$ ) and some $\text{snRNA}$ .
RNA Polymerase III: Located in the nucleus; transcribes transfer RNA ( $\text{tRNA}$ ), $5S\,tRNA$ , and some $\text{snRNA}$ .

Promoter Elements:
Core Promoter Elements: Located near the start site; specify where transcription begins.
TATA box (Hogness box): Located at $-30$ to $-25$ . The full consensus sequence is $5'\text{-TATAAAA-}3'$ .
Proximal Promoter Elements: Located between $-50$ and $-200$ ; required for high levels of transcription (e.g., CAAT box at $-70$ to $-80$ ).

The Transcription Complex:
TATA Binding Protein (TBP): A $34\,kDa$ protein that binds to the TATA box.
TFIID: A complex formed by TBP and several TBP-associated factors (TAFs). Binding of TFIID is the first step in forming the "committed complex."
The entire complex spans DNA from position $-30$ to $+30$ relative to the initiation site.

Enhancers and Silencers:
Cis-acting: Enhancers located on the same strand of DNA as the promoter.
Trans-acting: Enhancers acting on promoter elements on a different DNA strand.
Activators: Proteins that bring enhancers close to the promoter to achieve maximal transcription.

5' Capping:
Structure: A $7\text{-methylguanosine}$ cap is added to the $5'$ end of the mRNA via a $5'\text{-to-}5'$ triphosphate bridge.
Occurs in the nucleus before transport to the cytoplasm.
Functions: Protects mRNA from exonucleases and is required for efficient translation initiation.

3' Polyadenylation (Poly-A Tail):
Mechanism: mRNA is cleaved $10\text{-}35$ nucleotides downstream from the $AAUAAA$ recognition site (approximately $20\,bp$ from the site).
Enzyme: Poly(A) polymerase adds up to $200\,A$ residues.
Function: Protects mRNA from $5'$ exonucleases [per transcript note].

Intron Splicing:
Introns: Intervening sequences that do not code for proteins; must be removed in the nucleus.
Exons: The protein-coding sequences.
Spliceosome: The machinery for splicing, consisting of five $\text{snRNA}$ molecules ( $U1$ , $U2$ , $U4$ , $U5$ , and $U6$ ) and over $60$ proteins.
snRNP: Small ribonucleoprotein particles, often called "snurps," which facilitate splicing by positioning RNA segments and binding to consensus sequences at exon-intron junctions.
Splicing Consensus Sequences: - $5'\text{ Exon junction}$ : $5'\text{-AGGUAAGU-}3'$ - $3'\text{ Exon junction}$ : $5'\text{-CAGG-}3'$
Final Structure of Mature mRNA:
Includes the $5'\text{ CAP}$ , $5'\text{-UTR}$ (untranslated region), the Open Reading Frame (ORF) (starting with $AUG$ and ending with stop codons $UAA$ , $UAG$ , or $UGA$ ), the $3'\text{-UTR}$ , and the $poly(A)$ tail bound by the cap-binding complex and PAB (poly-A binding protein).