Translation is the process where mRNA codons are used to produce protein 11/28 and 11/30

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Translation is the process where mRNA codons are
used to produce protein 11/28 and 11/30

• What are the basics of translation?
• What are the 64 codons? Why only 61 tRNAs?
• How do eukaryotic and prokaryotic mRNAs differ?
• How does eukaryotic and prokaryotic translation
  differ?
• What are ribosomes composed of?
• What are the 4 ribosomal binding sites?
• How is tRNA charged with an amino acid?
• How is translation initiated
• What sets the reading frame of the codons in
  mRNA?
• How does peptide chain elongation occur?
• What terminates mRNA translation?

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Transcription Review how did we make mRNA?The
DNA duplex is unwound by RNAP to create access to
the template strand for transcription into a
complementary RNA sequence. NTPs are ONLY added
(elongation) to the RNA 3 end
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Translation is the process whereby the mRNA
sequence is converted into a protein sequence.

• Eukaryotic and Prokaryotic requirements are very
  similar!
• 1) Ribosome Large subunit and Small subunit
  (Svedberg Units)
• 2) tRNAs are required to carry the 20 different
  amino acids
• 3) mRNA must have a start sequence (AUG) and a
  stop codon
• mRNA characteristics
• Euk mRNA is modified with a 5-CH3 cap and
  3polyA-tail
• Prok mRNA has a special ribosomal binding site
  just upstream of AUG
• Shine Delgano Sequence critical for initiation to
  occur.
• Three Main Processes to Translation of mRNA
• 1) Initiation mRNA Bound by ribosome, then tRNA
  binds AUG
• 2) Elongation amino acids brought to new peptide
  by tRNAs
• 3) Termination peptide and ribosome released
  from mRNA

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The first 5A-U-G-3 encountered after the 5 end
of the mRNA is the start codon. Each triplet
of bases after this is a specific codon.

• There are 20 standard amino acids
• There are 4X4X464 codons
• Base triplet is a codon
• 61 codons are for amino acids
• 3 codons terminate translation
• AUG codes for methionine
• AUG Codes for Start
• Termination occurs with
• UAA UAG and UGA
• FrameShift Mutations change
• protein sequence by changing codon triplet
  identities on mRNA template
• CUGAUGA ?Add A
• CAUGAUGA ? new protein
• Position if 3rd nucleotide is the most variable
  Wobble

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Ribosomes are massive assemblies of proteins and
pieces of rRNA that form discrete small and
large subunits. Prokaryotes50S30S70S
Eukaryotes60S40S80S

• Ribosomes work free in the cytosol or when
  membrane bound
• i.e. Rough Endoplasmic Reticulum (RER)
• Small ribosomal subunit bind mRNA first, then
  Large subunit
• Complex has four RNA-lined binding sites
• 1) mRNA-Site
• 2) A-Site (aminoacyl-tRNA)
• 3) P-Site (peptidyl-tRNA
• 4) E-Site empty tRNA exits from here!
• We call a ribosome a ribozyme because it has
  enzyme-like activity but it is made of RNA at its
  active site, not protein!
• The Proteins associated with it help regulate its
  shape and activity but are not entirely critical
  for its activity

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The four binding sites on the ribosome are
located deep inside the complex and the sites
straddle the small and large subunits.Ribosomes
consists of 2/3 rRNA and 1/3 Protein by weight.
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tRNAs must be charged or activated by having
their specific amino acid added to the hydroxyl
located on their 3 end by a special enzyme
called aminoacyl-tRNA synthetase before
translation can progress.

• Some amino acids have several different codons
• There are 20 different amino acids
• Each amino acid type has a specific
  aminoacyl-tRNA synthetase that recognizes all
  codons associate with that specific amino acid
• There are 20 different kinds of aminoacyl-tRNA
  synthetase
• AAATPtRNA?aminoacyl-tRNA(charged)AMP 2 Pi
• Charging costs two Pi-Pi bonds! Expensive
• tRNA Anticodon binds complementary mRNA Codons
• mRNA 5-AUG-3 binds 3-UAC-5 of tRNA
• RNARNA binding is not as stable as DNA Double
  helix
• Wobble refers to the fact that the bases in the
  codon (especially the third) can be variable.

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How is the translation of mRNA to protein
initiated?

• Four Steps to Initiation
• 1) Small ribosomal subunit binds 5 end of mRNA
• 2) met-tRNA binds ribosome and crawls along mRNA
  until an AUG is encountered
• 3) Small subunit binds AUG and then attracts the
  large ribosomal subunit
• 4) Large subunit binds to small unit/mRNA to
  complete the ribosomal complex
• Initiation is now completed!
• Note
• The first AUG encountered by the ribosome sets
  the Reading Frame for all base-triplets
  (codons) that come after it, mRNA is read 3 bases
  at a time.
• A single mRNA can have several ribosomes on it at
  one time
• In prokaryotes a Shine-Delgano Sequence of
  AGGA- must precede the AUG for initiation (not
  required in eukaryotes)
• Prokaryotic Shine-Delgano sequence on mRNA
  5-AGGA-3 binds 3-UCCA-5 on the complementary
  rRNA stabilizing initiation complex.
• In prokaryotes the first amino acid is always
  formyl-methionine
• fMet gt Methionine with a formyl group on its
  amino end to make it non-reactive while being
  created

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Elongation additional charged tRNAs bring
additional amino acids to the open A-site on the
ribosome. The new aminoacyl-tRNA accepts the old
peptide chain. It becomes the P-site and the old
tRNA shifts into the E-site. Peptide elongation
process uses a ribozyme called
peptidyltransferase.
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Termination of translation occurs when a stop
codon UAC, UAG or AGA is exposed at the A-site.
Stop codon causes a release factor (protein) to
bind this open site causing the nascent peptide
to be released from the 3-end of tRNA at the
P-site.After release, the ribosome and mRNA
also dissociate.
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• Start Codon AUG Termination Codons UAC UAG and
  AGA
• 1) Given this mRNA how many amino acids long is
  the protein?
• 5UUCGAUG-GCC-UCU-UGC-AUG-GCG-UAG-UUU-AG3
• 1 5 10
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• 2) If Base 5 (A) is removed a new reading frame
  is established, where does synthesis start?
• 5UUCGUGGCCUCUUGC-AUG-GCG-UAG-UUU-AG.3
• 1 5 10 go
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• 3) If an extra base (G) is ADDED immediately
  after 7 the total mRNA becomes 31 bases, how
  many amino acids are produced?
• 5UUCGAUG-GGC-CUC-UUG-CAU-GGC-GUA-GUU-UGC...3
• 5go 10
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• Continue this by completing the assignment due
• Monday April 26th in class.10 points