Gene Transcription and RNA Modification: Study Notes

Gene Transcription and RNA Modification

Introduction to Transcription

Transcription is the process by which a DNA sequence is copied into an RNA sequence. This is the first step of gene expression, allowing genetic information stored in DNA to be used in the synthesis of proteins. The structure of DNA remains unchanged during transcription, ensuring the preservation of genetic information.

• Gene Expression: For protein-coding genes (structural genes), transcription produces messenger RNA (mRNA), which determines the amino acid sequence of a polypeptide during translation.

• Central Dogma: The flow of genetic information follows: DNA → RNA → Protein.

• Key Steps: DNA replication, transcription (DNA to RNA), and translation (RNA to protein).

Organization of Bacterial Genes and mRNA

Bacterial genes are organized with specific DNA sequences that control transcription and translation.

• Regulatory Elements: DNA sequences that control gene expression.

• Promoter: Site where RNA polymerase binds to initiate transcription.

• Terminator: Sequence signaling the end of transcription.

• mRNA Features: Ribosome-binding site, start codon, coding region (codons), and stop codon.

• Polycistronic mRNA: In bacteria, a single mRNA may encode multiple polypeptides.

Stages of Transcription

Transcription occurs in three main stages:

1. Initiation: RNA polymerase binds to the promoter, and the DNA is unwound to form an open complex.

2. Elongation: RNA polymerase synthesizes the RNA transcript by adding nucleotides in the 5' to 3' direction.

3. Termination: RNA polymerase and the RNA transcript dissociate from the DNA at the terminator sequence.

Transcription in Bacteria

Bacterial transcription has been extensively studied, especially in Escherichia coli (E. coli).

• Promoter Structure: Contains -35 and -10 consensus sequences (e.g., TTGACA at -35, TATAAT at -10).

• Transcriptional Start Site: Marked as +1, where RNA synthesis begins.

• RNA Polymerase: Composed of a core enzyme (α2, β, β', ω) and a sigma (σ) factor. The sigma factor is required for promoter recognition.

• Initiation: Sigma factor recognizes promoter, forms closed complex, then open complex as DNA unwinds.

• Elongation: RNA polymerase synthesizes RNA using the template (antisense) strand. The coding (sense) strand matches the RNA sequence (except T/U).

• Termination: Two mechanisms:

  • Rho-dependent: Requires the rho protein to separate the RNA-DNA hybrid.

  • Rho-independent: Relies on a stem-loop structure and a U-rich sequence in the RNA to cause dissociation.

Transcription in Eukaryotes

Eukaryotic transcription is more complex due to cellular compartmentalization and chromatin structure.

• Location: Occurs in the nucleus and is not coupled to translation.

• Chromatin Remodeling: Required to access DNA wrapped around histones.

• RNA Processing: Eukaryotic mRNAs undergo capping, polyadenylation, and splicing.

• RNA Polymerases:

  • RNA Pol I: Transcribes rRNA genes (except 5S rRNA).

  • RNA Pol II: Transcribes protein-coding genes (mRNAs) and some snRNAs.

  • RNA Pol III: Transcribes tRNA, 5S rRNA, and microRNA genes.

• Promoter Structure: Core promoter includes the TATA box (around -25) and transcriptional start site (+1). Regulatory elements (enhancers, silencers) modulate transcription.

• Transcription Factors: General transcription factors (GTFs) and mediator complex are required for RNA Pol II to initiate transcription.

General Transcription Factors (GTFs)

• TFIID: Recognizes the TATA box (contains TBP and TAFs).

• TFIIA: Assists TFIID binding.

• TFIIB: Binds to TFIID and recruits RNA Pol II.

• TFIIF: Binds to RNA Pol II and helps recruit TFIIE and TFIIH.

• TFIIE: Assists in open complex formation and TFIIH function.

• TFIIH: Acts as a helicase to unwind DNA and phosphorylates the CTD of RNA Pol II.

Termination in Eukaryotes

• Allosteric Model: RNA Pol II becomes destabilized after transcribing the polyadenylation signal and dissociates.

• Torpedo Model: An exonuclease degrades the RNA downstream of the cleavage site, causing RNA Pol II to dissociate.

RNA Modification

After transcription, RNA molecules undergo several modifications to become functional.

• 5' Capping: Addition of a 7-methylguanosine cap to the 5' end of mRNA. Occurs early during transcription and involves three steps:

  1. Removal of a phosphate from the 5' end.

  2. Addition of GMP via a 5'-5' linkage.

  3. Methylation of the guanine at the 7th position.

  Functions: Facilitates nuclear export, translation initiation, and splicing.

• 3' Polyadenylation: Addition of a polyA tail (string of adenines) to the 3' end of mRNA after cleavage near the AAUAAA signal. Increases mRNA stability and translation efficiency.

• RNA Editing: Chemical modification of bases (e.g., cytosine to uracil, adenosine to inosine) by deamination, altering the RNA sequence post-transcriptionally.

• Base Modification: Covalent modification of bases, especially in tRNAs (e.g., methylation).

Processing of rRNA and tRNA

• rRNA Processing: Ribosomal RNA is transcribed as a large precursor (e.g., 45S in eukaryotes) and cleaved into mature rRNAs (18S, 5.8S, 28S).

• tRNA Processing: Precursor tRNAs are cleaved at both ends by exonucleases and endonucleases (e.g., RNase P, a ribozyme). The CCA sequence is added to the 3' end, and introns are removed by splicing. Bases are often modified (e.g., methylguanosine, pseudouridine).

RNA Splicing

Splicing removes introns (non-coding regions) from pre-mRNA and joins exons (coding regions) to form mature mRNA.

• Splicing Mechanisms:

  • Group I Introns: Self-splicing using a free guanosine as a cofactor.

  • Group II Introns: Self-splicing using an internal adenosine's 2'-OH group, forming a lariat structure.

  • Spliceosome-mediated Splicing: Involves small nuclear ribonucleoproteins (snRNPs) that recognize specific sequences at exon-intron boundaries and catalyze splicing.

• Consensus Sequences:

  • 5' Splice Site: A/C GGU Pu AGUA

  • Branch Site: UACUUAUCC (with a critical adenosine)

  • 3' Splice Site: Py12 N Py AG G

Alternative Splicing

Alternative splicing allows a single gene to produce multiple mRNA variants, increasing protein diversity.

• Constitutive Exons: Always included in mature mRNA.

• Alternative Exons: Included or excluded depending on cell type or conditions.

• Regulation: Controlled by splicing factors (repressors and enhancers), such as SR proteins.

• Example: Alpha-tropomyosin gene produces different mRNAs in smooth vs. striated muscle cells by including different sets of exons.

Comparison of Bacterial and Eukaryotic Transcription and RNA Modification

Feature	Bacteria	Eukaryotes
Promoter	-35 and -10 sequences	TATA box and transcriptional start site (core promoter)
RNA Polymerase	Single RNA polymerase	Three types (I, II, III); Pol II for mRNA
Initiation	Sigma factor required	Five general transcription factors required
Elongation	Release of sigma factor	Mediator complex controls switch to elongation
Termination	Rho-dependent or rho-independent	Allosteric or torpedo model
RNA Processing	Rare (some rRNA, tRNA processing)	Extensive (capping, polyadenylation, splicing)

Summary Table of RNA Modifications

Modification	Description	Occurrence
Processing	Cleavage of large RNA into smaller functional pieces	rRNA, tRNA (prokaryotes and eukaryotes)
Splicing	Removal of introns and joining of exons	Pre-mRNA (eukaryotes), some rRNA, tRNA, rare in bacteria
5' Capping	Addition of 7-methylguanosine to 5' end	mRNA (eukaryotes)
3' Polyadenylation	Addition of polyA tail to 3' end	mRNA (eukaryotes), rare in bacteria
RNA Editing	Base sequence changed post-transcriptionally	Occasional in eukaryotes
Base Modification	Covalent modification of bases	tRNA (prokaryotes and eukaryotes)

Key Terms and Definitions

• Promoter: DNA sequence where RNA polymerase binds to initiate transcription.

• Terminator: DNA sequence signaling the end of transcription.

• Exon: Coding region retained in mature mRNA.

• Intron: Non-coding region removed during splicing.

• snRNP: Small nuclear ribonucleoprotein, component of the spliceosome.

• Spliceosome: Complex responsible for splicing pre-mRNA.

• Ribozyme: RNA molecule with catalytic activity.

• PolyA Tail: String of adenines added to the 3' end of mRNA.

• 5' Cap: 7-methylguanosine structure added to the 5' end of mRNA.

Key Equations and Chemical Reactions

• RNA Synthesis Direction: $\text{RNA is synthesized in the 5' \rightarrow 3' direction}$

• Base Pairing Rule in Transcription: $\text{A (DNA) \rightarrow U (RNA)}$ $\text{T (DNA) \rightarrow A (RNA)}$ $\text{G (DNA) \rightarrow C (RNA)}$ $\text{C (DNA) \rightarrow G (RNA)}$

• 5' Capping Reaction: $\text{pppN (5' end of RNA) + GTP \rightarrow G(5')ppp(5')N + PP_i}$ $\text{G(5')ppp(5')N + S-adenosylmethionine \rightarrow m^7G(5')ppp(5')N + S-adenosylhomocysteine}$

• Polyadenylation: $\text{pre-mRNA + nATP \xrightarrow{polyA\ polymerase} pre-mRNA-(A)_n + nPP_i}$

• RNA Editing (Cytosine Deamination): $\text{Cytosine + H_2O \xrightarrow{cytidine\ deaminase} Uracil + NH_3}$

Examples and Applications

• Alternative Splicing: The alpha-tropomyosin gene produces different protein isoforms in smooth and striated muscle cells by including different exons in the mature mRNA.

• Self-Splicing Introns: Group I introns in Tetrahymena rRNA genes can remove themselves without protein enzymes.

• RNA Editing: In mammals, apolipoprotein B mRNA is edited to produce two different proteins in the liver and intestine.

Additional info: Some details, such as the precise chemical structures of modified bases and the full list of snRNP functions, are omitted for brevity but can be found in advanced molecular biology texts.