Transcription and Post-Transcriptional Modifications

Transcription

Overview

  • Transcription is the process of creating RNA from a DNA template.
  • It is a crucial step in the central dogma of molecular biology (DNA → RNA).

RNAs produced during transcription:

  • Messenger RNA (mRNA): Carries genetic information from DNA to ribosomes for protein synthesis.
  • Transfer RNA (tRNA): Involved in carrying amino acids to the ribosome during protein synthesis.
  • Ribosomal RNA (rRNA): A structural component of ribosomes.
  • Other RNAs are also produced during transcription.
  • The primary location for transcription within the cell is the nucleus, where DNA is located.

Requirements for Transcription

  • DNA template/gene of interest: The specific DNA sequence to be transcribed.
  • Ribonucleoside triphosphates (rNTPs): Building blocks of RNA, including:
    • ATP (adenosine triphosphate)
    • GTP (guanosine triphosphate)
    • CTP (cytidine triphosphate)
    • UTP (uridine triphosphate) - Note the use of uracil (U) in RNA instead of thymine (T) in DNA.
  • RNA polymerase: The enzyme responsible for catalyzing RNA synthesis.

RNA Polymerases

  • RNA polymerase I: Produces most rRNA.
  • RNA polymerase II: Produces mRNA (the focus of this lecture).
  • RNA polymerase III: Produces tRNA.

Phases of Transcription

  • Assembly and Initiation
  • Elongation
  • Termination

Assembly and Initiation

  • Initiation Complex Formation:
    • Promoter: The region on DNA where transcription begins.
      • TATA box (proximal element/core promoter): A key promoter sequence where RNA polymerase II binds.
    • Distal elements (CAT box and GC box): Located upstream of the TATA box and control the frequency of transcription of the gene.
    • Enhancers and silencers: Located upstream and regulate transcription.
  • Transcription factors are also involved.
  • Unzipping the DNA to initiate access to the strand.

Elongation

  • Requires the initiation complex.
  • RNA polymerase II binds to the initiation site.
  • Ribonucleotide triphosphates are assembled to create the RNA transcript.
  • Occurs in the 5' to 3' direction (new nucleotides are added to the 3' end).
  • No primer is required for RNA polymerase (unlike DNA replication).
  • The gene is read, and the RNA transcript is formed.

Termination

  • Transcription stops when the termination sequence (TAA) is encountered.
  • This causes RNA polymerase and the pre-messenger RNA to detach. The termination sequence (TAA) causes the polymerase to fall off along with the pre-mRNA

Post-Transcriptional Modifications

  • Pre-messenger RNA is modified to produce the final messenger RNA transcript.
  • These modifications include:
    • 5' capping
    • Polyadenylation
    • Splicing

Modifications

  • Consider a completed transcript containing both introns and exons
5' Capping
  • A 7-methylguanosine cap is added to the 5' end.
  • Protects the mRNA from degradation by exonucleases.
Polyadenylation
  • A poly(A) tail (string of adenine bases) is added to the 3' end.
  • Also protects from degradation.
Splicing

  • Introns (non-coding regions) are removed, and exons (coding regions) are joined together.

  • Spliceosome: A complex that mediates splicing, associated with snRNPs.

    • snRNPs (small nuclear ribonucleoproteins): Made up of small nuclear RNA (snRNA) and proteins.

  • The spliceosome identifies consensus sequences at the ends of introns (GU at the beginning and AG at the end). These help identify target areas for splicing.

  • Introns are removed and stay in the nucleus.

  • Exons are joined to form the final mRNA.

  • The final mRNA (containing the 5' cap, exons, and poly(A) tail) leaves the nucleus for translation.