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.