Overview
This lecture focuses on the differences between prokaryotic and eukaryotic transcription and details the processing steps that convert pre-messenger RNA (pre-mRNA) to mature mRNA in eukaryotes.
Prokaryotic vs. Eukaryotic Transcription
- Eukaryotes have a membrane-bound nucleus, so transcription occurs in the nucleus and mRNA is transported to the cytoplasm for translation.
- Prokaryotes lack a nucleus, allowing transcription and translation to occur simultaneously in the cytoplasm.
- Eukaryotic mRNA is typically monogenic (encodes one protein), while prokaryotic mRNA is usually polygenic (encodes multiple proteins per mRNA).
- Eukaryotic cells use three RNA polymerases; prokaryotes use one.
- Eukaryotic mRNA is protected and lasts longer, while prokaryotic mRNA degrades rapidly.
Eukaryotic Transcription Mechanisms
- RNA Polymerase I transcribes rRNA genes; II transcribes protein-coding genes; III transcribes tRNA, some rRNA, and small nuclear RNA genes.
- Transcription factors (TFs) are required for RNA polymerases to bind DNA in eukaryotes.
- The eukaryotic promoter contains a TATA box located 25-35 bases upstream from the initiation site, recognized by transcription factors.
- Inserting a eukaryotic promoter before a bacterial gene in bacteria will not enable transcription due to promoter recognition differences.
Transcription Steps in Eukaryotes
- Initiation: Transcription factors and RNA polymerase II assemble at the promoter to form the initiation complex.
- Elongation: mRNA is synthesized 5' to 3' in the nucleus; FACT complex loosens and reforms nucleosomes for DNA access.
- Termination: RNA polymerase II transcribes beyond the gene; extra nucleotides are removed during mRNA processing.
mRNA Processing in Eukaryotes
- Pre-mRNA undergoes three modifications: intron splicing, addition of a 5’ methyl guanosine cap, and addition of a 3’ poly-A tail.
- Introns (non-coding sequences) are removed, and exons (coding sequences) are joined by spliceosomes.
- The 5’ cap and 3’ poly-A tail stabilize mRNA and prevent degradation.
Splicing and Spliceosome Function
- Spliceosome complexes, with proteins and small nuclear RNAs (snRNAs), catalyze intron removal and exon ligation.
- Spliceosome recognizes GU at the 5’ and AG at the 3’ end of introns.
- Mutations affecting splice sites or spliceosome components can cause faulty splicing and nonfunctional proteins.
rRNA and tRNA Processing & Function
- rRNA and tRNA are transcribed and processed but not translated; rRNA forms ribosomes, tRNA is involved in translation.
- tRNA has an amino acid attachment site and an anticodon region, which pairs with mRNA codons during translation.
- The amino acid attached to tRNA is determined by the mRNA codon, not the anticodon.
Key Terms & Definitions
- RNA polymerase — Enzyme that synthesizes RNA from a DNA template.
- Promoter — DNA sequence where transcription machinery assembles.
- TATA box — Consensus promoter sequence in eukaryotes, binding site for transcription factors.
- Intron — Non-coding region removed from pre-mRNA during processing.
- Exon — Coding region retained in mature mRNA.
- Spliceosome — Complex that removes introns from pre-mRNA.
- 5’ cap — Modified guanine nucleotide added to the 5’ end of mRNA for stability.
- Poly-A tail — String of adenine nucleotides added to the 3’ end of mRNA.
- Codon — Sequence of three mRNA nucleotides specifying an amino acid.
- Anticodon — tRNA sequence complementary to the mRNA codon.
Action Items / Next Steps
- Complete the RNA splicing animation linked on the Canvas website.
- Review the process of mRNA processing and splicing.
- Prepare for next lecture on translation (mRNA to protein).