Exam Notes on Telomeres, Central Dogma, and Transcription

Telomeres and Telomerase

  • The Problem: DNA polymerase cannot fill the gap left after RNA primer removal at the ends of linear chromosomes, leading to shortening with each replication cycle.
  • Solution: Eukaryotes use telomeres (tandemly repeated sequences) at chromosome ends.
  • Telomerase:
    • Composed of protein and RNA (complementary to the telomere repeat).
    • Binds to the telomere repeat and adds new repeats, lengthening the chromosome.
    • Absence or mutation leads to chromosome shortening and limited cell division.
  • Mechanism:
    • Telomerase RNA acts as a template for adding nucleotides to the 3' end of the G-rich strand.
    • After several nucleotides are added, the RNA template moves along the DNA.
    • Synthesis on the complementary strand fills the gap.

Central Dogma and Reverse Transcription

  • Central Dogma: DNA -> RNA -> Protein (Replication, Transcription, Translation).
  • Reverse Transcription: Some RNA viruses (retroviruses) use reverse transcriptase to make a DNA copy of their RNA.
    • The DNA copy (provirus) integrates into the host chromosome.
    • Retrotransposons are DNA sequences that make RNA copies of themselves, which are then reverse-transcribed back into DNA and inserted into new locations in the genome; retrotransposons remain within the cell.

Prions

  • Definition: Infectious proteins that cause diseases like mad cow disease and Creutzfeld-Jakob syndrome.
  • Mechanism:
    • Normal prion protein (PrPc) misfolds into PrPSc.
    • PrPSc converts normal PrPc into more PrPSc, spreading the disease; this is a form of inheritance not involving nucleic acids.

Transcription

  • Definition: Process of making an RNA copy of a gene.
  • Enzyme: RNA polymerase (e.g., RNA polymerase II in eukaryotes for most genes).
  • Raw Materials: Ribonucleoside triphosphates (ATP, CTP, GTP, UTP).
  • Direction: Proceeds 5' to 3', adding new bases to the 3' OH group.
  • Primer: Does not require a primer; starts at a promoter region.
  • Promoter: Located upstream (5') of the first transcribed base; has consensus sequences.
  • Requirements: Template (dsDNA), activated precursors, divalent metal ions (Mg2+, Mn2+), RNA polymerase enzyme.

RNA Polymerase

  • Function: Searches for promoter sites, unwinds DNA, selects correct ribonucleoside triphosphate, catalyzes phosphodiester bond formation, detects termination signals, interacts with transcription factors.
  • Holoenzyme (Prokaryotes):
    • Core enzyme (α₂ßß'): carries out the polymerization.
    • Sigma (σ) factor: recognizes promoter sites.
    • The holoenzyme is essential for initiation at the correct start site.

Transcription Steps

  • Initiation: RNA polymerase binds to the promoter with the help of transcription factors.
  • Elongation: RNA polymerase unwinds DNA and synthesizes an RNA copy.
  • Termination:
    • Prokaryotes: Ends at a specific terminator sequence.
    • Eukaryotes: Transcription does not have a definite end point.

Termination Types

  • Rho-independent: mRNA forms a hairpin followed by U residues, causing polymerase to stall and separate.
  • Rho-dependent: Rho (ρ) helicase binds to a rut site on the RNA, migrates to the polymerase, and separates the mRNA.

Eukaryotic vs. Prokaryotic Promoters

  • Prokaryotic: -10 (Pribnow box: TATAAT) and -35 elements (TTGACA).
  • Eukaryotic: More diverse; includes TATA box, initiator elements, GC box, CAAT box.
    • TATA box binds TATA-binding protein (TBP), which is a subunit of TFIID.
    • TFIIH separates DNA strands to give the RNA Polymerase access to single-stranded DNA template.

Eukaryotic RNA Polymerases

  • RNA polymerase I: Located in the nucleolus; transcribes most rRNAs.
  • RNA polymerase II: Located in the nucleus; transcribes protein-coding pre-mRNAs, miRNAs, and lncRNAs
  • RNA polymerase III: Located in the nucleus; transcribes 5S pre-rRNA, pre-tRNAs, and snRNAs.

Eukaryotic Termination

  • RNA Polymerase I: Terminates at a specific sequence recognized by TTF-1.
  • RNA Polymerase III: Terminates after a short stretch of U's.
  • RNA Polymerase II:
    • Transcribes past the end of the gene.
    • The transcript is cleaved at an internal site between an AAUAAA sequence and a GU-rich sequence.
    • CPSF binds AAUAAA and CstF binds the GU-rich sequence.
    • Poly(A) polymerase adds a poly-A tail.
    • The remaining transcript is digested by a 5'-exonuclease (Xrn2) until it catches up to RNA polymerase II, causing termination.

RNA Processing (Eukaryotes)

  • Steps:
    1. Add a 5' cap (7-methyl G).
    2. Add a 3' poly-A tail (100-200 A's).
    3. Splice out introns (using spliceosomes).
  • Introns: Removed from the primary RNA transcript in the nucleus; exons are re-attached.
    *After processing, the messenger RNA is exported out of the nucleus to cytoplasm for translation.