Transcription and translation

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Transcription and translation

• The link between genes and enzymes

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Inborn errors of metabolism

• A. Garrod, 1902
• Certain diseases seemed to be inherited
• Alkaptonuria due to an enzymatic defect
• Beadle and Tatum, 1941- one gene, one enzyme
• Studied Neurospora crassa
• Nutritional (auxotrophic) mutants
• Biosynthetic pathways

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Beadle and Tatum experiment

• Pathway for arginine biosynthesis was known
• Used media with defined supplements to identify
  mutants
• One gene, one polypeptide hypothesis defines
  relationship between DNA and protein

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The central dogma of molecular biology

• DNA
• ?
• RNA
• ?
• Protein

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RNA is involved in protein synthesis, too

• Messenger RNA- the gene sequence
• Ribosomal RNA- structural component of the
  ribosome
• Transfer RNA- interpret mRNA and build the amino
  acid sequence on the ribosome
• In eukaryotes
• Small nuclear RNAs-
• SRPs (signal recognition particles)
• miRNA (micro-RNA)

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The triplet code

• Four nucleotides
• Triplets give 64 possible combinations to code
  for 20 amino acids
• Code is nonoverlapping
• One DNA strand serves as the template

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Transcription RNA copied from DNA

• RNA polymerase uses DNA as a template to make RNA
• Template is the antisense strand
• 5-TACGGTACATTCGTACC ATC T -3
• 3-ATGCCATGTAAGCATGGTAGA -5
• mRNA5- UACGGUACAUUCGUACCAUCU-3

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Deciphering the genetic code

• 64 possible codons
• Experimental requirements
• Cell-free systems with all the necessary enzymes
• Synthetic RNAs
• Nirenberg, 1961-6
• Worked with different combinations of RNAs to
  deduce codons
• Khorana (overlapped Nirenberg)
• Synthesized RNA molecules of defined sequences
  and analyzed peptides produced
• Also synthesized acetyl CoA and the first
  artificial chromosome (1970)

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(No Transcript)
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Some exceptions to the code

• Genetic code is shared by all organisms
• Some variations in mitochondrial, chloroplast and
  protozoan DNA (ribosomes are different, too)
• Processes of transcription and translation are
  very similar in prokaryotes and eukaryotes, but
  not identical
• Initiation, elongation, termination

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Properties of RNA polymerase

• Two forms holoenzyme and core polymerase
• Need both for accurate initiation of RNA
  synthesis
• Promoter and start site
• Accurate termination
• Process is simpler in prokaryotes one polymerase

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Models of transcription events
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Operons transcription and translation coupled in
prokaryotes
repressor
Regulatory promoter
operator structural genes region
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Eukaryotic vs prokaryotic transcription

• Eukaryotes have three RNA polymerases, each with
  their own promoters
• RNA polymerase I- rRNA
• Probably species-specific
• RNA polymerase II- mRNA and snRNAs
• Core promoter
• RNA polymerase III- tRNA and some small RNAs
• Promoter is internal
• All work in cell nucleus

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Eukaryotic initiation complex contains many
cofactors
Note that transcription factors bind first
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Elongation and termination of transcription

• Multiple molecules of polymerase can transcribe
  DNA simultaneously
• Termination mechanism is different in eukaryotes

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Another difference posttranscriptional
modification

• 5 cap
• Guanine is methylated and linked to 5 end of
  transcript
• 3 poly-A tail
• Specific cleavage site AAUAA
• Poly-A polymerase adds poly-A tail after that
  site
• Splicing of pre-mRNA

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Eukaryotic mRNA is spliced

• Introns and exons
• Much more human DNA is in introns than exons
  (exons are 1-1.5 of total)
• Splicing involves snRNPs

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Model for pre-mRNA splicing
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What are the rules for intron frequency and size?

• There are none
• Some genes have many introns, some none
• Probably accumulated over time
• Exon shuffling seen among some families of
  proteins
• Alternative splicing seen (remember the codon
  rule)

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Overview of translation
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Structure of transfer RNA (tRNA)
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tRNA charging ensures that amino acids are
positioned correctly
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tRNA and the ribosome

• E site- for tRNA with amino acid already added
  (exit)
• P site- for amino acid being added (peptidyl)
• A site for the incoming amino acid (aminoacyl)
• This is where peptide bonds are formed (remember
  primary structure?)

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Initiation of translation in prokaryotesnote
the order of events
AUG is start codon first amino acid is
N-formylmethionine Methionine in eukaryotes also
more initiation factors Small subunit binds to
5-cap instead of a ribosome-binding sequence
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Elongation cycle
Wobble pairing gives flexibility (note
orientation of anticodon)
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Termination triggered by a stop codon
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In eukaryotes, many proteins are processed in the
ER

• Prokaryotes have this mechanism, too
• Secreted proteins require it

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Summary gene expression in bacteria
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Gene expression in eukaryotes
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Mutations occur in DNA
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Does mutation affect phenotype?

• Triplet repeats- add to reading frame but do not
  shorten it
• Might be in coding or noncoding region
• Sometimes they are big enough to see in a
  karyotype

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Chromosomal mutations can have drastic effects

• Deletions
• Duplications
• Can lead to gene families and pseudogenes
• Inversions
• Translocations
• Can be fatal, can be inherited tend to arise in
  single cells (somatic mutation)

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Summary

• Genes specify polypeptides
• Transcription makes RNA copies of DNA
• Translation involves mRNA, tRNA, and rRNA in
  protein synthesis
• Processes are similar in prokaryotes and
  eukaryotes, but there are significant differences
• The genetic code is nearly universal
• Mutations alter DNA and can alter genes and
  proteins
• Evolution arises from mutation