7' Expression of Genetic Information 3

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7. Expression of Genetic Information (3)
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Encoding Genetic Information

• The Genetic Code
• 20 different aa
• 4 different nucleotides
• Requires at least a range of combinations of 3
  nucleotides 43 gives 64 possible combinations
• If 64 combinations specify 20 aa
• What is the function of the remaining 44 codes
• Some aa are specified by more than one codon
• A degenerative code

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Encoding Genetic Information

• The Genetic Code
• The code is highly degenerate
• Nearly all codes specify aas
• Those that do not are stop codons (3 of the 64)
• Cause reading of the message to stop
• For all organisms the same codons specify the
  same aas
• Exception codons of mitochondrial mRNAs

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Encoding Genetic Information

• The Genetic Code

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Encoding Genetic Information

• The Genetic Code
• Chart shows aa assignments
• Non-random
• Tend to be clustered
• Reflects similar codons specifying the same aa
• Spontaneous mutations causing a single base
  change
• May not cause an aa change
• Similar aa are specified by similar codons
• Greatest similarities in first two nucleotides
• eg glycine 4 codons all GGX
• Greatest variability in third nucleotide of the
  triplet

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Encoding Genetic Information

• The Genetic Code

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Encoding Genetic Information

• Decoding Transfer RNAs
• tRNAs act like adaptors
• Each tRNA
• linked to a specific aa
• Able to recognize a particular codon of mRNA

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Encoding Genetic Information

• Decoding Transfer RNAs Structure
• All 73 to 93 nucleotides
• Unusual bases
• Enzymatic modification of bases after
  incorporation into the tRNA chain
  posttranscriptionally
• Structure disrupts H-bonding
• Recognition sites for proteins in loop structures
• Strings of complementary sequences
• Folded into double strand structure
• In 2 dimensions appears as a clover leaf
• aa is attached to the 3 adenine

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Encoding Genetic Information

• Decoding Transfer RNAs Structure
• tRNAs fold into a defined tertiary structure
• L shape
• Each has unique features

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Encoding Genetic Information

• Decoding Transfer RNAs Structure
• tRNA mRNA complementary base pairing
  facilitates translation
• Interacting tRNA domain
• Three nucleotides termed the anticodon
• Located in the middle loop
• Loop contains seven nucleotides anticodon
  middle three
• Opposite end of molecule from aa attachment

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Encoding Genetic Information

• Decoding Transfer RNAs
• mRNA codons
• first two nucleotides greatest similarities
• Third nucleotide greatest variation
• Crick proposed the wobble hypothesis
• Same tRNA recognizes more than one codon
• Rules of wobble at third position
• U of anticodon pairs with A or G of mRNA
• G of anticodon pairs with U or C of mRNA
• I (inosine) pairs with U, C or A of mRNA

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Encoding Genetic Information

• Decoding Transfer RNAs aa activation
• aas are covalently linked at the 3 end of tRNA
• Enzyme aminoacyl-tRNA synthase
• Each aa recognized by a specific aminoacyl-tRNA
  synthase
• Aminoacyl-tRNA synthases two step reaction
• ATP aa
  aminoacyl-AMP PPi
• Aminoacyl-AMP tRNA aminoacyl-tRNA AMP

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Encoding Genetic Information

• Decoding Transfer RNAs aa activation
• Aminoacyl-tRNA synthases two step reaction
• ATP aa aminoacyl-AMP PPi
• Aminoacyl-AMP tRNA aminoacyl-tRNA AMP

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Encoding Genetic Information

• Translating genetic information
• Most complex synthetic activity in the cell
• Translation in bacterial cells
• Similar in Eukaryotic cells
• Difference translation in eukaryotic cells a
  larger number of soluble (non-ribosomal) protein
  factors
• Synthesis three distinct activities
• Initiation
• Elongation
• Termination

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Encoding Genetic Information

• Translating genetic information
• Initiation
• Ribosome moves along mRNA from one codon to next
• To ensure proper triplets are read
• Ribosome attaches at a precise site the
  initiation codon
• AUG
• Ribosome locked into proper reading frame
• Mechanism described in a series of steps ---

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Encoding Genetic Information

• Translating genetic information
• Initiation
• Step 1 Small ribosomal subunit initiation
  codon interaction
• Binding of small ribosomal subunit to first AUG
• Bacterial mRNAs a specific sequence of
  nucleotides
• Shine-Delgarno sequence
• 5 to 10 nucleotides before initiation sequence
• Complementary to a sequence of nucleotides near
  the 3 end of bacterial small subunit

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Encoding Genetic Information

• Translating genetic information
• Initiation
• Step 1 Small ribosomal subunit initiation
  codon interaction
• Attachment via this interaction in complementary
  sequences
• Initiation factors also involved

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Encoding Genetic Information

• Translating genetic information
• Initiation
• Step 2 first aa-tRNA brought to ribosome
• AUG also codes for methionine
• Always the first aa
• Two methionyl-tRNAs
• Initiator of protein synthesis tRNAiMet
• General methionyl t-RNA tRNAMet
• tRNAiMet enters complex by binding to AUG and
  initiation factor (IF2)

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Encoding Genetic Information

• Translating genetic information
• Initiation
• Step 3 Assembling initiation complex
• Large ribosomal subunit joins the complex
• GTP bound to IF2 is hydrolyzed
• Release of IF2-GDP

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Encoding Genetic Information

• Translating genetic information
• Role of the ribosome
• A molecular motor (kinesin and dynein)
• During translation
• Repetitive cycle of mechanical changes
• Driven by energy release of GTP hydrolysis
• Ribosomal RNAs play a major role in selecting
  tRNAs
• Accurate translation
• Polymerization of aa

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Encoding Genetic Information

• Translating genetic information
• Role of the ribosome
• Ribosome has three sites for association with
  tRNAs
• A site aminoacyl site
• P site peptidyl site
• E site exit site
• tRNAs bind these sites gap between ribosomal
  subunits

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Encoding Genetic Information

• Translating genetic information
• Role of the ribosome

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Encoding Genetic Information

• Translating genetic information
• Role of the ribosome
• Interface contains binding sites for mRNA and
  incoming tRNA
• Catalytic portion of large subunit in a deep
  cleft hydrophobic
• Tunnel through large subunit translocation of
  peptide
• RNA associated proteins stabilize the tertiary
  structure

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Encoding Genetic Information

• Translating genetic information
• Elongation
• Step 1 Aminoacyl-tRNA selection
• tRNAiMet is in place at the P site
• A site is available for entry of the next
  aa-tRNA
• Before binding of the aa-tRNA to the ribosome -
  it must first bind to a protein elongation factor
  - EF-Tu
• EF-Tu is GTP-linked
• EF-Tu delivers the aa-tRNA to the ribosomal A
  binding site

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Encoding Genetic Information

• Translating genetic information
• Elongation
• Step 2 Peptide bond formation
• At end of step 1- aa-tRNA 1 and 2 juxtaposed
  for reaction
• Amino group of aa at the A site reacts with the
  carboxyl group of the aa at the P site
• Peptide bond formation occurs spontaneously (no
  energy input)
• Catalysed by peptidyl transferase
• Component of the large ribosomal subunit
• Peptidyl transferase is a ribozyme

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Encoding Genetic Information

• Translating genetic information
• Elongation
• Step 3 Translocation
• Following formation of first peptide bond tRNA
  at the A site is bound to a dipeptide and to
  mRNA
• The tRNA on the P site is devoid of an aa
• In translocation the ribosome and mRNA move
  relatively
• Ribosome moves 3 nucleotides (one codon) along
  mRNA in the 5 to 3 direction
• Accompanied by movement of the tRNA dipeptide
  from the A to the P site
• The deacylated tRNA moves from the P site to the
  E site
• Translocation promoted by a GTP-bound elongation
  factor (EF-G in prokaryotes, eEF2 in eukaryotes)

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Encoding Genetic Information

• Translating genetic information
• Elongation
• Step 4 Release of deacylated tRNA
• Deacylated tRNA leaves the ribosome emptying
  the E site
• Each cycle of elongation uses 2 GTP
• 1 in aminoacyl tRNA selection
• 1 in translocation
• Once peptidyl-tRNA has moved to the P site the
  A site is again vacant and ready for entry of
  another aminoacyl-tRNA

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Encoding Genetic Information

• Translating genetic information
• Elongation

Step 1
Step 2
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Encoding Genetic Information

• Translating genetic information
• Elongation

Step 3
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Encoding Genetic Information

• Translating genetic information
• Termination
• No tRNAs exist whose anticodons are complementary
  to a stop codon
• mRNA stop codons UAA, UAG and UGA
• Signal is read to stop further elongation and
  release the polypeptide associated with the last
  tRNA
• Termination requires the presence of release
  factors
• Bacteria have 3 RF1, RF2 and RF3
• Eukaryotes have 2 eRF1 and eRF3
• Work together to recognize all stop codons
• An example of molecular mimicry
• Release factor proteins resemble a tRNA

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Encoding Genetic Information

• Translating genetic information
• Termination
• Release factors enter the A site
• A tripeptide in the release factor substitutes
  for the anticodon of tRNA and interacts directly
  with the stop codon
• Release factor 3 carries a bound GTP which is
  hydrolysed
• Once translation stops
• Peptide severed from attachment to last tRNA in
  the P site
• Both release factor and deacylated tRNA are
  released from the ribosome
• Ribosome then separates from mRNA and dissociates
  into small and large subunits

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Encoding Genetic Information

• Translating genetic information
• Termination

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Encoding Genetic Information

• Translating genetic information
• Termination

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Encoding Genetic Information

• Translating genetic information - Overview

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Encoding Genetic Information

• Translating genetic information
• Polyribosomes
• During translation multiple ribosomes are
  attached along the mRNA thread
• Complex is a polyribosome or polysome
• Each ribosome assembles at the initiation codon
• Moves toward the 3 end of mRNA
• Simultaneous translation greatly increases the
  rate of protein synthesis

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Encoding Genetic Information

• Translating genetic information