Transcription and the Genetic Code

Comparison of DNA and RNA

Sugar component: DNA contains deoxyribose; RNA contains ribose, which has a hydroxyl group at the $2'$ carbon ( $2'\text{-OH}$ ).
Nitrogenous bases: DNA uses adenine (A), guanine (G), cytosine (C), and thymine (T); RNA substitutes uracil (U) for thymine.
Strandedness: DNA exists as a double-stranded helix; RNA is predominantly single-stranded.
Biological persistence and enzymatic activity: DNA is chemically stable and maintains protein-encoding instructions; RNA is short-lived, regulates genetic expression, and can function catalytically as an enzyme (ribozyme).

Bacterial Transcription Machinery and Promoters

RNA polymerase enzyme complex:
- Core enzyme ( $\alpha_2\beta\beta'\omega$ ):
  - $\alpha \times 2$ : Assembles the enzyme and interacts with regulatory proteins.
  - $\beta$ : Catalytic site for ribonucleotide polymerization.
  - $\beta'$ : Binds and holds the non-template DNA strand.
  - $\omega$ : Facilitates assembly and stabilizes the $\beta'$ subunit.
- Holoenzyme: Formed by the core enzyme plus a regulatory sigma ( $\sigma$ ) factor (e.g., $\sigma^{70}$ ), which confers promoter recognition and initiation specificity.
Polymerization reaction:
- Synthesizes RNA in the $5' \rightarrow 3'$ direction from a $3' \rightarrow 5'$ DNA template strand using ribonucleoside triphosphates (NTPs: ATP, UTP, CTP, GTP): $n(\text{NTP}) \xrightarrow{\text{DNA, RNA polymerase}} (\text{NMP})_n + n(\text{PP}_i)$
- Driven forward by the exergonic hydrolysis of inorganic pyrophosphate ( $\text{PP}_i$ ).
- Requires no primer to initiate synthesis de novo.
Consensus promoter elements:
- $-10$ region (Pribnow box): Consensus sequence $\text{TATAAT}$ .
- $-35$ region: Consensus sequence $\text{TTGACA}$ .
- Transcription start site is designated as $+1$ .

Bacterial transcription initiation, elongation, and sigma subunit dissociation

Stages of Bacterial Transcription

Initiation:
- Holoenzyme binds promoter DNA to establish a closed promoter complex.
- DNA unwinds to produce an open complex containing a $10\text{--}12\,\text{bp}$ transcription bubble.
- Generates short abortive transcripts up to $8\,\text{nt}$ before achieving promoter escape after synthesizing approximately $11\,\text{nt}$ .
- $\sigma$ subunit dissociates from the core enzyme upon entering processive elongation.
Elongation:
- The core enzyme traverses the template at roughly $20\,\text{bases/s}$ , unwinding approximately $10\,\text{bp}$ at a time.
- Uses a single active site for both ribonucleotide polymerization and proofreading (error removal).
Termination mechanisms:
- Rho-independent (intrinsic) termination (~ $80\%$ ):
  - Transcription of an inverted repeat generates an RNA hairpin loop that stalls RNA polymerase.
  - Stalling over an adjacent poly-U tract results in unstable $A\text{-}U$ hybrid base pairing, causing the transcript and polymerase to release from DNA.
- Rho-dependent termination (~ $20\%$ ):
  - Nascent RNA contains a rut (rho utilization) site bound by Rho ( $\rho$ ), a hexameric helicase.
  - Rho translocates along RNA in the $5' \rightarrow 3'$ direction and catches up to the polymerase while it is paused at an inverted-repeat hairpin, unwinding the RNA-DNA hybrid.

Eukaryotic Transcription and Chromatin Architecture

Chromatin structure:
- Nucleosome core consists of an octamer containing two copies each of histones H2A, H2B, H3, and H4 wrapped by $147\,\text{bp}$ of DNA ( $6\,\text{nm} \times 11\,\text{nm}$ disc).
- Histone H1 acts as a linker histone to compact nucleosomes into a $30\text{-nm}$ fiber.
- Histones are rich in positively charged lysine and arginine residues, enabling electrostatic interactions with negatively charged DNA phosphate backbones.
- Unstructured, protruding histone tails undergo covalent modifications during chromatin remodeling to expose promoter DNA to trans-acting factors.
Classes of eukaryotic RNA polymerases:
- RNA Polymerase I: Located in the nucleolus; transcribes ribosomal RNA (rRNA).
- RNA Polymerase II: Located in the nucleoplasm; transcribes pre-mRNA, small nuclear RNA (snRNA), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs).
- RNA Polymerase III: Located in the nucleoplasm; transcribes $5\text{S}\,\text{rRNA}$ and transfer RNA (tRNA).

Eukaryotic Initiation and Pre-Initiation Complex Formation

Promoter elements:
- Core promoter: Contains the TATA box (Goldberg-Hogness box) positioned around $-30$ relative to $+1$ (consensus $\text{TATA(A/T)AAR}$ , where R is a purine).
- Proximal promoter elements: Located upstream near $-70$ .
- Distal regulatory elements: Enhancers and silencers located upstream or downstream, often exceeding $1000\,\text{bp}$ from the core promoter.
Pre-Initiation Complex (PIC) assembly:
- TFIID (containing TATA-binding protein, TBP) binds directly to the TATA box.
- TFIIA and TFIIB bind to stabilize the TFIID-DNA complex.
- RNA Polymerase II is recruited in association with TFIIF.
- TFIIE and TFIIH join the complex; TFIIH functions as a helicase to unwind DNA and as a kinase to phosphorylate the C-terminal domain (CTD) of RNA Pol II, triggering promoter clearance.

Assembly of the eukaryotic pre-initiation complex at the TATA box

Eukaryotic Termination and mRNA Processing

$5'$ Capping:
- Addition of $7\text{-methylguanosine}$ ( $m^7\text{G}$ ) to the $5'$ end via an unusual $5'\text{-to-}5'$ triphosphate bridge.
- Prevents exonucleolytic degradation and serves as a ribosomal recognition signal for translation initiation.
Cleavage and Polyadenylation:
- Pre-mRNA contains an $\text{AAUAAA}$ polyadenylation signal upstream and a $\text{GU/U-rich}$ element downstream.
- Cleavage factors (CPSF, CstF, CFI, CFII) cleave the transcript between these signals.
- Poly-A polymerase (PAP) adds $100\text{--}300$ (up to $500$ ) non-templated adenine residues to generate the $3'$ poly-A tail, bound and stabilized by PAB II.
Torpedo termination:
- Following poly-A site cleavage, the downstream RNA undergoes co-transcriptional cleavage (CoTC), creating an uncapped $5'$ monophosphate end.
- Xrn2 ( $5' \rightarrow 3'$ exonuclease) degrades the uncapped RNA until it reaches RNA Pol II, dislodging the enzyme from the DNA template.

RNA Splicing and Transcript Modification

Spliceosome-mediated splicing:
- Removes intervening sequences (introns) and ligates expressed regions (exons; composed of the $5'\text{ UTR}$ , protein coding sequence [CDS], and $3'\text{ UTR}$ ).
- Consensus sequences: $5'$ splice donor site ( $\text{GU}$ ), branch point adenosine ( $\text{A}$ ), and $3'$ splice acceptor site ( $\text{AG}$ ).
- Carried out by snRNPs (U1, U2, U4, U5, U6).
- Two-step transesterification:
  1. The $2'\text{-OH}$ of the branch point A attacks the $5'$ splice donor phosphate, forming a branched lariat intermediate.
  2. The free $3'\text{-OH}$ of the upstream exon attacks the $3'$ splice acceptor phosphate, joining the exons and releasing the excised lariat.

Spliceosome assembly, lariat formation, and exon ligation during pre-mRNA splicing

Self-splicing introns (Ribozymes):
- Group I: Present in bacteria, lower eukaryotes, and higher plants; uses the $3'\text{-OH}$ of an exogenous guanosine cofactor ( $\text{exoG}$ ) to initiate cleavage at the $5'$ splice site.
- Group II: Present in fungal and plant organelles; uses an internal branch point A residue to form a lariat intermediate without protein spliceosomes.
Alternative splicing:
- Differential exon inclusion or intron retention produces diverse mature mRNAs and protein isoforms from a single pre-mRNA.
- Occurs in approximately $95\%$ of human multi-exon genes (e.g., tropomyosin).
RNA editing:
- Post-transcriptional nucleotide alteration independent of the genomic DNA sequence.
- Cytidine deamination (APOBEC-1): Converts C to U; changes a glutamine codon ( $\text{CAA}$ ) to a stop codon ( $\text{UAA}$ ) in ApoB pre-mRNA, producing truncated ApoB-48.
- Adenosine deamination (ADAR): Converts A to I (inosine, paired as G); alters glutamine to arginine in GluR channel transcripts, modifying $Ca^{2+}$ permeability in the central nervous system.

The Genetic Code

Fundamental properties:
- Triplet format: Composed of $64$ codons specifying $20$ amino acids ( $61$ coding codons, $1$ start codon [ $\text{AUG}$ , Methionine], and $3$ stop codons [ $\text{UAA}$ , $\text{UAG}$ , $\text{UGA}$ ]).
- Commaless and non-overlapping: Translated linearly without punctuation inside an open reading frame (ORF), though viruses (e.g., $\phi\text{X174}$ ) can contain overlapping reading frames.
- Degenerate and ordered: Multiple codons specify the same amino acid, with chemically similar amino acids grouped by related middle-base codons.
Wobble hypothesis:
- Non-standard base pairing occurs between the third base ( $3'$ end) of the mRNA codon and the first base ( $5'$ end) of the tRNA anticodon:
  - Anticodon A pairs with codon U.
  - Anticodon C pairs with codon G.
  - Anticodon G pairs with codon C or U.
  - Anticodon U pairs with codon A or G.
  - Anticodon I (inosine) pairs with codon A, U, or C.
Exceptions to the universal code:
- Human and yeast mitochondria: $\text{UGA}$ specifies Trp instead of termination.
- Human mitochondria: $\text{AUA}$ specifies Met instead of Ile; $\text{AGA}$ and $\text{AGG}$ serve as stop codons instead of Arg.
- Yeast mitochondria: $\text{CUA}$ specifies Thr instead of Leu.
- Ciliate protozoa (Paramecium, Tetrahymena, Stylonychia): $\text{UAA}$ and $\text{UAG}$ encode Gln instead of terminating translation.