Overview
This lecture covers DNA transcription, focusing on differences between prokaryotic and eukaryotic cells, the process of transcription, gene regulation, post-transcriptional modifications, and related diseases.
Definition and Purpose of Transcription
- Transcription is the process of converting DNA into RNA.
- Both prokaryotic and eukaryotic cells perform transcription but with different enzymes and factors.
Transcription in Prokaryotes
- Prokaryotes use one RNA polymerase holoenzyme made of a core enzyme (2 alpha, beta, beta', omega subunits) and a sigma subunit.
- The core enzyme reads DNA and synthesizes RNA; the sigma subunit binds the enzyme to the promoter region.
- One RNA polymerase synthesizes all types of RNA (mRNA, tRNA, rRNA).
Transcription in Eukaryotes
- Three different RNA polymerases:
- RNA polymerase I makes rRNA,
- RNA polymerase II makes mRNA and snRNA,
- RNA polymerase III makes tRNA and some snRNA/rRNA.
- General transcription factors are required for RNA polymerase binding.
- Promoter regions include TATA box, CAAT box, and GC box.
Gene Regulation in Eukaryotes
- Gene expression is modulated by enhancers (increase transcription) and silencers (decrease transcription).
- Specific transcription factors bind to enhancers/silencers causing DNA looping, facilitating or inhibiting transcription.
Stages of Transcription
Initiation
- In prokaryotes, RNA polymerase holoenzyme binds to promoter (-35, -10/Pribnow box, +1 start site).
- In eukaryotes, RNA polymerase II and general transcription factors (esp. TFII D) bind to the promoter region (e.g., TATA box).
Elongation
- RNA polymerases read the template (antisense) DNA strand 3’→5’ and synthesize RNA 5’→3’.
- No strong proofreading function is confirmed for RNA polymerases.
Termination
- In prokaryotes:
- Rho-dependent requires Rho protein to detach RNA polymerase,
- Rho-independent uses hairpin loop from inverted repeats to trigger release.
- In eukaryotes: Polyadenylation signal (AAUAAA) signals enzyme cleavage and termination.
Post-transcriptional Modification (Eukaryotes Only)
- hnRNA (heterogeneous nuclear RNA) undergoes modifications to become mature mRNA.
- 5’ capping adds 7-methylguanosine for protection and translation initiation.
- 3’ poly-A tail added by polyA polymerase for stability and export.
- Splicing removes non-coding introns and joins exons, performed by snRNPs ("snurps").
- Alternative splicing can produce multiple protein variants from one gene.
- RNA editing can alter nucleotide sequence, e.g., CAA to UAA, changing protein product (apoB100 to apoB48).
Key Terms & Definitions
- Transcription — DNA to RNA synthesis.
- Promoter — DNA sequence where transcription starts.
- RNA Polymerase — Enzyme synthesizing RNA from DNA.
- Sigma Factor — Subunit allowing prokaryotic RNA polymerase to bind promoter.
- General Transcription Factors — Proteins aiding eukaryotic RNA polymerase promoter binding.
- Enhancer/Silencer — DNA elements increasing/decreasing transcription.
- Exon — Coding RNA sequence for protein.
- Intron — Non-coding sequence spliced out.
- snRNP ("snurp") — Small nuclear ribonucleoprotein complex, mediates splicing.
- 5’ Cap — Modified guanine nucleotide added to mRNA for stability.
- Poly-A Tail — String of adenines at mRNA 3’ end for stability.
- Alternative Splicing — Process generating different mRNAs from same gene.
- RNA Editing — Enzymatic alteration of RNA sequence after synthesis.
Action Items / Next Steps
- Review differences in transcription between prokaryotes and eukaryotes.
- Memorize functions of RNA polymerases I, II, III.
- Practice sketching and labeling promoter regions and transcription stages.
- Read about diseases related to splicing errors (e.g., spinal muscular atrophy, beta thalassemia).