Transcription and Post-Transcriptional Modification

Transcription

• DNA contains the code for proteins, but protein synthesis happens in the cytoplasm.
• DNA can't leave the nucleus because it would be degraded. RNA is used to transmit genetic information.
• Transcription: mRNA creation from a DNA template.
• mRNA carries information directly from DNA.
• tRNA and rRNA are other types of RNA that play roles in protein translation.

Mechanism of Transcription

• Transcription copies only one of the two DNA strands.
• Initiation:
  • Enzymes like helicase and topoisomerase unwind DNA, preventing supercoils.
  • This allows access to the gene of interest.
• Transcription synthesizes a single mRNA strand from the DNA template (antisense strand).
• The mRNA strand is antiparallel and complementary to the DNA template strand.
• RNA is synthesized by DNA-dependent RNA polymerase.
• RNA polymerase finds genes by searching for promoter regions.
• Eukaryotes:
  • RNA polymerase II transcribes mRNA.
  • The TATA box (high concentration of thymine and adenine) is the binding site in the promoter region.
  • Transcription factors help RNA polymerase locate and bind to the promoter.
• RNA polymerase, unlike DNA polymerase, doesn't need a primer.
• Eukaryotes have three RNA polymerases, but only one transcribes mRNA.
  • RNA polymerase I: Located in the nucleolus, synthesizes rRNA.
  • RNA polymerase II: Located in the nucleus, synthesizes hnRNA (preprocessed mRNA) and some snRNA.
  • RNA polymerase III: Located in the nucleus, synthesizes tRNA and some rRNA.
• RNA polymerase moves along the template strand in the 3' to 5' direction.
  • This allows mRNA construction in the 5' to 3' direction.
• RNA polymerase doesn't proofread, so the transcript isn't edited.
• The coding (sense) strand isn't used as a template but is identical to the mRNA (except T is replaced by U).
• Numbering system:
  • The first base transcribed to RNA is +1.
  • Bases upstream (5' end) are negative numbers (-1, -2, -3, etc.).
  • Bases downstream (3' end) are positive numbers (+2, +3, +4, etc.).
  • No nucleotide is numbered zero.
• The TATA box is usually around -25.
• Transcription continues until the RNA polymerase reaches a termination sequence.
• The initial transcript is hnRNA (heterogeneous nuclear RNA).
• mRNA is derived from hnRNA via post-transcriptional modification.

Post-Transcriptional Processing

• hnRNA must undergo processing to interact with ribosomes and survive in the cytoplasm.
• Maturation includes:
  • Splicing (introns and exons).
  • 5' cap.
  • 3' poly-A tail.

Splicing Introns and Exons

• Non-coding sequences (introns) are removed, and coding sequences (exons) are linked.
• Splicing is done by the spliceosome.
  • snRNA molecules couple with proteins (snRNPs).
  • The snRNP/snRNA complex identifies the 5' and 3' splice sites of introns.
  • Introns are excised in a lariat shape and degraded.
• The function of introns is not fully understood.
  • Hypotheses:
    • Regulation of gene expression.
    • Maintaining genome size.
    • Allowing rapid protein evolution.
    • Modular function: standard sequences swapped in/out.

Five Cap

• A 7-methylguanylate triphosphate cap is added to the 5' end of hnRNA during transcription.
• The cap is recognized by the ribosome as a binding site.
• It also protects mRNA from degradation.

Three Poly A Tail

• A poly(A) tail (adenine bases) is added to the 3' end.
• It protects against rapid degradation.
• The longer the tail, the longer the mRNA survives.
• It also assists with export from the nucleus.
• After processing, only exons remain, and the cap and tail are added, creating mature mRNA.
• Untranslated regions (UTRs) remain at the 5' and 3' ends.
• Ribosome initiates translation at the start codon AUG and ends at a stop codon (UAA, UGA, or UAG).

Alternative Splicing

• hnRNA can be spliced in different ways to produce protein variants.
• This allows more proteins from a limited number of genes.
• Humans make around 100,000 proteins from about 20,000-25,000 genes.
• Alternative splicing also regulates gene expression.