6.3 Transcription & RNA Processing

Transcription is the regulated process by which genomic DNA sequences are copied into complementary RNA molecules. In eukaryotes, pre-messenger RNA (pre-mRNA) undergoes complex post-transcriptional modifications before nuclear export and translation.

Transcription Mechanism & Polarity

RNA synthesis is catalyzed by RNA Polymerases, which read a single-stranded DNA template in the $3' \rightarrow 5'$ direction to synthesize RNA strictly in the $5' \rightarrow 3'$ direction.

Template (Antisense) vs. Coding (Sense) Strands

• Antisense (Template) Strand: The DNA strand transcribed by RNA polymerase. Its sequence is antiparallel and complementary to the synthesized RNA transcript.
• Sense (Coding) Strand: The non-template DNA strand. Its sequence and $5' \rightarrow 3'$ polarity are identical to the resulting RNA transcript, with the single exception that Thymine (T) in DNA is replaced by Uracil (U) in RNA.

Eukaryotic RNA Polymerases (RMT Mnemonic)

Eukaryotes utilize three distinct nuclear RNA polymerases, classified by their transcript specificity and sensitivity to $\alpha$-amanitin (a cyclic peptide toxin from the death cap mushroom Amanita phalloides):

1. RNA Polymerase I: Located in the nucleolus. Synthesizes the single $45\text{S}$ pre-rRNA precursor, which is processed into $28\text{S}$, $18\text{S}$, and $5.8\text{S}$ rRNAs. Insensitive to $\alpha$-amanitin.
2. RNA Polymerase II: Located in the nucleoplasm. Synthesizes pre-messenger RNA (pre-mRNA), microRNAs (miRNAs), and snRNAs. Extremely sensitive to low concentrations of $\alpha$-amanitin (which binds Pol II, inhibiting translocation along DNA and causing massive hepatic necrosis).
3. RNA Polymerase III: Located in the nucleoplasm. Synthesizes transfer RNAs (tRNAs), $5\text{S}$ rRNA (the only rRNA made outside the nucleolus), and U6 snRNA. Sensitive to high concentrations of $\alpha$-amanitin.

Prokaryotic Comparison: Bacteria utilize a single RNA polymerase core enzyme ($\alpha_2\beta\beta'\omega$) combined with a transient $\sigma$ (sigma) factor to form the holoenzyme responsible for promoter recognition.

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Transcriptional Regulation

Promoters & Pre-Initiation Complex

Promoters are cis-acting DNA sequences immediately upstream of the transcription start site ($+1$).

• Eukaryotes: The core promoter contains the TATA box (Hogness box), located at position $-25$ to $-30$ relative to $+1$ (consensus sequence $5'-TATAAA-3'$). The TATA box is bound by the TATA-binding protein (TBP) subunit of general transcription factor TFIID. Additional general transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH) assemble to form the Pre-Initiation Complex (PIC). TFIIH possesses helicase activity to unwind promoter DNA and kinase activity to phosphorylate the C-terminal domain (CTD) of RNA Pol II, initiating elongation.
• Prokaryotes: Core promoters feature the Pribnow box ($-10$ region, consensus $5'-TATAAT-3'$) and the $-35$ sequence ($5'-TTGACA-3'$), recognized by $\sigma^{70}$.

Enhancers & Silencers

• Enhancers: Cis-acting regulatory DNA sequences that bind specific activator transcription factors to stimulate transcription. Enhancers function independently of distance, position, or orientation relative to the promoter (located thousands of base pairs upstream, downstream, or within introns).
• Silencers: Cis-acting DNA sequences that bind repressor proteins to suppress transcription.
• DNA Looping Mechanism: Activators bound to distant enhancers physically contact general transcription factors at the promoter through DNA looping, facilitated by architectural proteins (HMGA) and the multi-protein Mediator complex.

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Eukaryotic Post-Transcriptional Pre-mRNA Processing

Nascent pre-mRNA undergoes three mandatory nuclear processing steps before translation:

Nascent pre-mRNA ---> [5' Capping (m7G)] ---> [3' Polyadenylation (Poly-A Tail)] 
                 ---> [Spliceosomal Intron Excision] ---> Mature mRNA Export

1. 5' Capping

Co-transcriptionally, when pre-mRNA reaches ~20–30 nucleotides in length, an enzyme complex adds a 7-methylguanosine ($m^7G$) cap to the $5'$ end via an unusual $5'-to-5'$ triphosphate linkage.

• Functions:
  1. Protects pre-mRNA from degradation by $5' \rightarrow 3'$ exoribonucleases (e.g., XRN1).
  2. Serves as a recognition signal for nuclear export and eukaryotic initiation factor 4E (eIF4E) during translation initiation.

2. 3' Polyadenylation

Transcription continues past the coding region until RNA Pol II transcribes the polyadenylation signal sequence ($5'-AAUAAA-3'$).

• Cleavage & Addition: Cleavage factors cleave pre-mRNA ~10–30 nucleotides downstream of the signal sequence. Poly-A Polymerase (PAP) adds ~200–250 Adenine residues (poly(A) tail) to the $3'-OH$ terminus in a template-independent manner using ATP.
• Functions:
  1. Protects the $3'$ transcript end from $3' \rightarrow 5'$ exoribonucleases.
  2. Binds Poly(A)-Binding Protein (PABP) to promote nuclear export and enhance translation efficiency by circularizing mRNA via interactions with eIF4G.

3. Pre-mRNA Splicing

Splicing removes non-coding introns and covalently ligates coding exons. Splicing is executed by the Spliceosome, a massive ribonucleoprotein complex comprising five small nuclear ribonucleoproteins (snRNPs: U1, U2, U4, U5, U6) and auxiliary proteins.

Conserved Intronic Sequences

• 5' Splice Site: Invariant $5'-GU-3'$ sequence at the 5' intron boundary.
• 3' Splice Site: Invariant $5'-AG-3'$ sequence at the 3' intron boundary.
• Branch Point Adenine: An internal Adenine residue located 18–40 nucleotides upstream of the 3' splice site.

Two-Step Transesterification Mechanism

1. Step 1: The $2'-OH$ group of the internal Branch Point Adenine performs a nucleophilic attack on the phosphate of the invariant $GU$ at the 5' splice site. This cleaves the 5' exon-intron junction, creating an unusual $2'-5'$ phosphodiester bond that forms a lariat intermediate structure.
2. Step 2: The free $3'-OH$ group of the cleaved 5' exon performs a nucleophilic attack on the phosphate of the invariant $AG$ at the 3' splice site. This covalently ligates the 5' and 3' exons together and releases the excised intron as a lariat, which is rapidly degraded by debranching enzymes.

Step 1: Branch Point 2'-OH ---> 5' Splice Site (GU) ---> Lariat Intermediate
Step 2: 5' Exon 3'-OH    ---> 3' Splice Site (AG) ---> Exon Ligation + Intron Lariat

Alternative Splicing & Proteomic Diversity

Alternative Splicing selectively includes or excludes specific exons or introns from mature mRNA. Regulated by cis-acting enhancers/silencers and trans-acting splicing factors (SR proteins, hnRNPs), alternative splicing permits a single gene to encode multiple distinct protein isoforms with differing functions, tissue distributions, or cellular localizations (e.g., membrane-bound vs. secreted IgM antibodies). Alternative splicing explains how ~20,000 human protein-coding genes generate $>100,000$ unique cellular proteins.