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Lecture 7-3
 

Lecture 7-3

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    Lecture 7-3 Lecture 7-3 Presentation Transcript

    • 7. Expression of Genetic Information (3)
      • The Genetic Code
        • 20 different 
        • 4 different nucleotides
        • Requires at least a range of combinations of 3 nucleotides – 4 3 – gives 64 possible combinations
        • If 64 combinations specify 20 
          • What is the function of the remaining 44 codes
        • Some  are specified by more than one codon
        • A degenerative code
      Encoding Genetic Information
      • The Genetic Code
        • The code is highly degenerate
        • Nearly all codes specify  s
        • 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  s
          • Exception – codons of mitochondrial mRNAs
      Encoding Genetic Information
      • The Genetic Code
      Encoding Genetic Information
      • The Genetic Code
        • Chart shows  assignments
          • Non-random
          • Tend to be clustered
            • Reflects similar codons specifying the same 
        • Spontaneous mutations causing a single base change
          • May not cause an  change
        • Similar  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
      Encoding Genetic Information
      • The Genetic Code
      Encoding Genetic Information
      • Decoding – Transfer RNAs
        • tRNAs act like adaptors
        • Each tRNA
          • linked to a specific 
          • Able to recognize a particular codon of mRNA
      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’
        •  is attached to the 3’ adenine
      Encoding Genetic Information
      • Decoding – Transfer RNAs – Structure
        • tRNAs fold into a defined tertiary structure
          • L shape
          • Each has unique features
      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  attachment
      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
      Encoding Genetic Information
      • Decoding – Transfer RNAs –  activation
        •  s are covalently linked at the 3’ end of tRNA
          • Enzyme – aminoacyl-tRNA synthase
          • Each  recognized by a specific aminoacyl-tRNA synthase
        • Aminoacyl-tRNA synthases – two step reaction:
        • ATP +  aminoacyl-AMP + PP i
        • Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP
      Encoding Genetic Information
      • Decoding – Transfer RNAs –  activation
        • Aminoacyl-tRNA synthases – two step reaction:
        • ATP +  aminoacyl-AMP + PP i
        • Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP
      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
      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 ---
      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
      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
      Encoding Genetic Information
      • Translating genetic information
        • Initiation
          • Step 2 – first  -tRNA brought to ribosome
            • AUG also codes for methionine
            • Always the first 
            • Two methionyl-tRNAs
              • Initiator of protein synthesis – tRNA i Met
              • General methionyl t-RNA – tRNA Met
            • tRNA i Met enters complex by binding to AUG and initiation factor (IF2)
      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
      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 
      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
      Encoding Genetic Information
      • Translating genetic information
        • Role of the ribosome
      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
      Encoding Genetic Information
      • Translating genetic information
        • Elongation
          • Step 1 – Aminoacyl-tRNA selection
            • tRNA i Met is in place at the P site
            • ‘ A’ site is available for entry of the next  -tRNA
              • Before binding of the  -tRNA to the ribosome - it must first bind to a protein elongation factor - EF-Tu
              • EF-Tu is GTP-linked
              • EF-Tu delivers the  -tRNA to the ribosomal A binding site
      Encoding Genetic Information
      • Translating genetic information
        • Elongation
          • Step 2 – Peptide bond formation
            • At end of step 1-  -tRNA #1 and #2 juxtaposed for reaction
            • Amino group of  at the A site reacts with the carboxyl group of the  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
      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 
            • 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)
      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
      Encoding Genetic Information
      • Translating genetic information
        • Elongation
      Encoding Genetic Information Step 1 Step 2
      • Translating genetic information
        • Elongation
      Encoding Genetic Information Step 3
      • 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
      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
      Encoding Genetic Information
      • Translating genetic information
        • Termination
      Encoding Genetic Information
      • Translating genetic information
        • Termination
      Encoding Genetic Information
      • Translating genetic information - Overview
      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
      Encoding Genetic Information
      • Translating genetic information
      Encoding Genetic Information