Gene Expression and Transcription

Gene Expression

• Gene expression is the process of converting DNA information into protein molecules
• Gene expression occurs in two steps: transcription and translation
• Transcription is the process of making an RNA copy of a gene sequence
• Translation is the process of translating the nucleotide sequence of an mRNA molecule to the amino acid sequence of a protein

Transcription

• Transcription is the first stage of gene expression
• In eukaryotic cells, transcription of mRNA involves:
  • Synthesis of RNA molecules (formation of the first transcript, pre-mRNA)
  • RNA processing/modifications (intron splicing, end modifications) to form mature mRNA
• RNA synthesis requires enzymes called RNA polymerases
• RNA polymerase was first discovered by J.Hurwitz and S.Weissman in 1960

RNA Types

• Messenger RNA (mRNA): used as a template for protein synthesis
• Ribosomal RNAs (rRNA):
• Transfer RNA (tRNA): functions in translation of mRNA to protein
• Small RNAs: function in mRNA splicing, rRNA processing, and regulation of gene expression

Transcription Requirements

• DNA template
• RNA polymerase
• Ribonucleoside triphosphates (ATP, GTP, CTP, UTP)
• Divalent metal ions (Magnesium (Mg+2) or Mn+2)

Stages of RNA Synthesis

• Binding of RNA polymerase to promoter sequences of DNA template
• Initiation of RNA synthesis
• Elongation of polynucleotide chain
• Termination of synthesis

Prokaryotic Transcription

• E. coli is a model organism for transcription studies
• mRNA was first discovered in E. coli
• RNA polymerase was purified from E. coli
• Mechanisms that regulate gene expression were discovered in E. coli

RNA Polymerase

• The enzyme responsible for RNA synthesis
• Requires DNA as a template and catalyzes the polymerization of ribonucleoside 5'-triphosphates (NTPs)
• Brings complementary bases together
• Always catalyzes RNA synthesis in 5' → 3' direction
• Does not need primers
• Synthesized RNA chain and template DNA strand are antiparallel to each other

Prokaryotic RNA Polymerase

• Consists of six polypeptides (2α, β, β', ω, and σ subunits)
• Sigma (σ) subunit recognizes promoter sequences on DNA
• When RNA reaches a few nucleotides in length, σ is separated from the other subunits

Bacterial RNA Polymerase

• Core RNA polymerase consists of 2α, 1β, 1β', and 1ω subunits
• Catalyzes step-by-step elongation of RNA chain
• Requires σ subunit for binding to specific DNA sequences (promoter regions)

Promoter Sequences

• Discovered in E. coli
• Consensus sequences contain 6 nucleotides
• Located upstream of transcription initiation sites
• Two distinct DNA sequences:
  • -10 region with consensus TATAAT
  • -35 region with consensus TTGACA (35 bp before initiation site)

Initiation of Transcription

• Binding of σ subunit of RNA polymerase to promoter regions (closed promoter complex)
• Opening and unwinding of small portion (14bp) of DNA double helix (-12 to +2) (open promoter complex)
• Transcription initiation by polymerization of first two ribonucleoside triphosphates

Elongation of RNA Chain

• RNA polymerase moves forward, synthesis continues
• RNA chain elongates
• During elongation, polymerase unwinds DNA ahead of it and rewinds DNA behind it
• Maintains an unwound region of about 15 base pairs in the region of transcription
• Internal channel between β and β' subunits of RNA polymerase contains 20 bp of DNA and forms the active site of the polymerase

Termination of Transcription

• RNA synthesis continues until RNA polymerase encounters the termination signal
• Transcription stops