General Biology/Genetics/Gene Expression

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Flow of genetic information

• DNA → mRNA → polypeptide
• Transcription: DNA → mRNA
  • RNA polymerase
  • Nucleus in eukaryotes
  • Transcription also makes rRNA and tRNA
• Translation: mRNA → polypeptide
  • Ribosomes: protein and rRNA
  • Genetic code and tRNA

The Genetic Code

• Triplet codon
  • 64 triplet codons (43)
  • Experimentally deciphered in 1961
• Nearly universal
  • Implies common ancestor to all living things
  • Minor exceptions: certain ciliates, mitochondria, chloroplasts
• Still evolving

Transcription

• RNA polymerase
  • NTP substrates
  • Synthesizes single stranded RNA complementary to template strand of DNA
  • New nucleotides to 3’ end
• Begins at promoter site
  • no primer necessary
• Ends at terminator site
• Much posttranscriptional modification in eukaryotes

Transcription bubble

Promoter site

• Prokaryotes
  • -10 nt, TATA box
  • -35 nt, additional signal
• Eukayotes
  • -25, TATAAA box
  • Additional signals upstream
• Promoters may be strong or weak
• In eukaryotes, access to promoter depends upon state of chromatin coiling

Eukaryote mRNA

• Synthesized as pre-mRNA, processed in nucleus
• 5’ end: GTP cap placed in inverted position
  • Essential for ribosome recognition
• 3’ end: poly-A tail; non-templated addition of ~50-250 A nucleotides; stability
• Introns: intervening sequences removed

Translation

• Requires:
  • mRNA
  • tRNA
  • ribosomes
  • translation factors (various proteins)
• In prokaryotes, takes place on growing mRNA
• In eukaryotes, in cytoplasm on free ribosomes and RER
• AUG start codon to stop codon

Translation in bacteria

tRNA

• Transfer RNA
• Two important parts
  • Anticodon
• Hydrogen bonds with mRNA codon
  • 3’ end
• Accepts amino acid (using energy of ATP)
• Aminoacyl-tRNA synthetase

Aminoacyl tRNA synthase

• Enzyme used to bind amino acid from the cytoplasm to tRNA, which then transfers that amino acid to the ribosome for polypeptide formation

Translation

• Initiation complex
  • Small ribosomal subunit
  • mRNA
  • fMet-tRNA (prokaryotes only; met-tRNA in eukaryotes)
  • Initiation factors
• Elongation
  • Ribosome
  • mRNA
  • tRNAs
  • Elongation factors

Elongation, translocation

• incoming tRNA enters the A site
• rRNA catalyzes peptide bond formation. Note that growing peptide is attached to what was incoming tRNA at P site after translocation.
• empty tRNA leaves via E site; recycled
• A site ready for next charged tRNA

Introns/exons

• In eukaryotes, coding regions of gene may be interrupted by introns, noncoding regions of DNA (RNA)
• Introns
  • 22- >10,000 nt in length
  • 5’ GU … 3’ AG removal sequence
  • Not essential to genes
  • May constitute >90% of gene
• removed from pre-mRNA to form mRNA
• Exon: often codes for functional domain of protein
  • translatable mRNA

References

This text is based on notes very generously donated by Paul Doerder, Ph.D., of the Cleveland State University.