Gene Expression: Transcription

Overview of Gene Expression

Gene expression is the process by which the information encoded in DNA is used to direct the synthesis of RNA and proteins. Transcription is the first step, where DNA serves as a template for RNA synthesis. The central dogma of molecular biology describes the directional flow of genetic information: DNA → RNA → Protein.

• Transcription: Synthesis of RNA from a DNA template; the nucleotide language remains the same.

• Translation: Synthesis of protein from RNA; the language changes from nucleotides to amino acids.

• Exceptions: RNA viruses can reverse transcribe RNA into DNA (reverse transcription), and some viruses replicate RNA from RNA templates.

Example: Retroviruses such as HIV use reverse transcriptase to synthesize DNA from their RNA genome.

18.1 The Directional Flow of Genetic Information

The Central Dogma and Its Refinements

The central dogma states that genetic information flows from DNA to RNA to protein. However, exceptions exist, such as reverse transcription in retroviruses and RNA replication in some viruses.

• Central Dogma: DNA → RNA → Protein

• Reverse Transcription: RNA → DNA (catalyzed by reverse transcriptase)

• RNA Replication: RNA → RNA (in some viruses)

Transcription and Translation: Prokaryotes vs. Eukaryotes

Cellular Locations and Coupling

Transcription and translation occur in different locations in prokaryotes and eukaryotes, affecting their regulation and efficiency.

• Prokaryotes: No nuclear envelope; transcription and translation are coupled. Translation can begin before transcription is complete, forming polyribosomes.

• Eukaryotes: Transcription occurs in the nucleus; translation occurs in the cytoplasm. Processes are compartmentalized and separated.

Example: In bacteria, multiple ribosomes translate a single mRNA simultaneously, increasing protein synthesis speed.

18.2 Mechanisms of Transcription

RNA Structure and Chemistry

RNA is similar to DNA but has ribose sugar, uracil instead of thymine, and is usually single-stranded.

• Ribose: Sugar in RNA; has a 2' hydroxyl group.

• Uracil: Replaces thymine in RNA.

• Single-stranded: Most RNA molecules are not double-stranded.

Stages of Transcription

Transcription consists of four main stages: binding, initiation, elongation, and termination.

1. Binding: RNA polymerase binds to a promoter sequence, unwinding the DNA locally.

2. Initiation: RNA polymerase begins RNA synthesis using one DNA strand as a template.

3. Elongation: RNA polymerase moves along the DNA, elongating the RNA chain in the 5' → 3' direction.

4. Termination: RNA polymerase dissociates from the DNA, releasing the RNA transcript.

Bacterial Transcription: Promoters and RNA Polymerase

Bacterial transcription involves specific promoter sequences and a multi-subunit RNA polymerase.

• Promoter: DNA sequence upstream of the transcription start site; includes the -10 (Pribnow box: TATAAT) and -35 (TTGACA) elements.

• Consensus Sequence: Most common nucleotides at each position in a promoter.

• UP Elements: Upstream sequences enhancing promoter strength, especially in rRNA genes.

RNA Polymerase Structure and Function

Bacterial RNA polymerase is composed of multiple subunits, including the sigma (σ) factor, which is essential for promoter recognition.

• Core Enzyme: α2ββ' (catalytic activity)

• Holoenzyme: Core enzyme + σ factor (promoter specificity)

• σ Factor: Promotes binding at -35 and -10 elements; different σ factors recognize different promoters.

Initiation and Elongation

RNA polymerase unwinds DNA and synthesizes RNA without a primer. Early abortive synthesis produces short RNA fragments until a stable transcript is formed.

• Scrunching: Polymerase pulls downstream DNA into its interior, creating a bulge.

• Elongation: RNA chain grows; DNA ahead is unwound, behind is rewound. Topoisomerases prevent supercoiling.

Proofreading and Termination

RNA polymerase has limited proofreading ability. Termination occurs via two mechanisms: intrinsic (hairpin loop) and rho-dependent.

• Intrinsic Termination: GC-rich hairpin followed by U's causes RNA release.

• Rho-dependent Termination: Rho factor binds a termination sequence and unwinds RNA from DNA.

Eukaryotic Transcription: Complexity and RNA Polymerases

Eukaryotic transcription is more complex, involving three distinct RNA polymerases and varied promoter structures.

• RNA Polymerase I: Synthesizes rRNA (except 5S); located in nucleolus; insensitive to α-amanitin.

• RNA Polymerase II: Synthesizes mRNA, snRNA, microRNA; located in nucleoplasm; highly sensitive to α-amanitin.

• RNA Polymerase III: Synthesizes tRNA, 5S rRNA, other small RNAs; located in nucleoplasm; moderately sensitive to α-amanitin.

Promoters and Transcription Factors in Eukaryotes

Eukaryotic promoters are diverse and require general transcription factors for RNA polymerase binding and initiation.

• Core Promoter: Minimal sequence required for transcription initiation; includes elements like the TATA box, initiator (Inr), BRE, and DPE.

• Upstream Control Elements: Enhance transcription efficiency; include CAAT box, GC box, and enhancers.

• General Transcription Factors: Proteins (e.g., TFIID, TFIIH) required for all transcription; TFIID contains TATA-binding protein (TBP).

RNA Synthesis, Termination, and Polyadenylation

Termination signals differ for each polymerase. RNA polymerase II transcripts are cleaved and polyadenylated at the 3' end.

• Poly(A) Tail: Added by poly(A) polymerase; protects mRNA and aids export and translation.

• Cleavage Site: Located 10–35 nucleotides downstream of AAUAAA signal.

18.3 RNA Processing and Turnover

RNA Processing: Maturation of Primary Transcripts

Primary RNA transcripts undergo processing to become mature, functional RNAs. This includes capping, polyadenylation, splicing, and chemical modifications.

• 5' Cap: Methylated guanosine added via 5'→5' linkage; stabilizes mRNA and aids translation initiation.

• Poly(A) Tail: 50–250 adenines added to 3' end; increases stability and facilitates export.

• Splicing: Removal of introns and joining of exons; catalyzed by spliceosomes.

Ribosomal RNA (rRNA) Processing

rRNA is synthesized as a precursor (pre-rRNA) and processed by cleavage and methylation, guided by small nucleolar RNAs (snoRNAs).

• Nucleolus: Site of rRNA synthesis and ribosome assembly.

• Nucleolus Organizer Region (NOR): Contains multiple rRNA gene copies.

• rRNA Types: 18S (small subunit), 28S, 5.8S, 5S (large subunit).

Transfer RNA (tRNA) Processing

tRNA molecules are synthesized as precursors and processed by removal of leader and trailer sequences, addition of CCA at the 3' end, and chemical modification of bases.

• Cloverleaf Structure: Secondary structure with four hairpin loops.

• Base Modifications: Methylation, creation of unusual bases (e.g., inosine).

• Intron Removal: Some tRNAs contain introns excised during processing.

Messenger RNA (mRNA) Processing in Eukaryotes

Eukaryotic mRNA undergoes capping, polyadenylation, and splicing. Introns are removed, and exons are joined to form mature mRNA.

• Heterogeneous Nuclear RNA (hnRNA): Mixture of mRNA and precursors in the nucleus.

• 5' Cap: Added soon after transcription initiation.

• Poly(A) Tail: Added after cleavage at the polyadenylation site.

• Splicing: Introns removed by spliceosomes; exons joined.

Introns, Exons, and Splicing

Introns are non-coding sequences removed from pre-mRNA; exons are coding sequences retained in mature mRNA. Splicing is catalyzed by spliceosomes, which recognize GU-AG boundaries and branch points.

• Spliceosome: Complex of snRNPs and proteins; assembles on pre-mRNA.

• GU-AG Rule: Most introns start with GU and end with AG.

• Lariat Structure: Formed during splicing; intron is excised as a loop.

• Exon Junction Complex (EJC): Deposited at exon-exon boundaries; aids export and regulation.

Self-Splicing Introns and Ribozymes

Some introns are self-splicing, acting as ribozymes. Group I and Group II introns are found in organelle genomes and some prokaryotes.

• Group I Introns: Excised as linear RNA fragments.

• Group II Introns: Excised as lariats; similar mechanism to spliceosomes.

• Ribozyme: RNA molecule with catalytic activity.

Alternative Splicing and Exon Shuffling

Alternative splicing allows a single gene to produce multiple protein products. Exon shuffling enables evolution of new proteins by recombination between introns.

• Alternative Splicing: Regulatory proteins and snoRNAs influence splice site selection.

• Exon Shuffling: Recombination between introns creates new exon combinations.

RNA Editing

RNA editing alters nucleotide sequences post-transcriptionally, affecting protein coding and function.

• Insertion/Deletion: Guide RNAs direct uracil addition/removal (e.g., trypanosome mitochondria).

• Base Conversion: C-to-U or A-to-I conversions in plant organelles and animal cells.

• MicroRNAs: Can be edited to alter regulatory functions.

Coordination of RNA Processing

The C-terminal domain (CTD) of RNA polymerase II coordinates RNA processing events, including capping, splicing, and polyadenylation, often occurring cotranscriptionally.

• CTD: Contains repeats of a seven-amino-acid sequence; phosphorylation triggers processing events.

Nuclear Export and mRNA Turnover

Mature mRNA is exported from the nucleus via the nuclear pore complex. Only properly processed transcripts are exported. mRNA turnover is measured by half-life, with eukaryotic mRNAs lasting hours to days, and bacterial mRNAs only minutes.

• Nuclear Export Factor 1 (NXF1): Interacts with cap-binding complex and EJC for export.

• mRNA Turnover: High turnover allows rapid regulation of gene expression.

Amplification of Genetic Information

Multiple mRNA copies can be synthesized from a single gene, allowing amplification of protein production.

• Example: Silkworm fibroin gene produces 104 mRNA copies per cell, each yielding 105 protein molecules.

Summary Table: Classes of Naturally Occurring RNA

Key Equations and Concepts

• Phosphodiester Bond Formation:

• Direction of RNA Synthesis:

• Consensus Sequence Example:

Additional info:

• RNA processing centers such as Cajal bodies, nuclear speckles, and PML bodies play roles in RNA maturation and chromatin organization.

• RNA editing and DNA editing are important for gene regulation and defense against viruses.