Protein Synthesis

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• Protein Synthesis

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Translating the Message

• How does the sequence of mRNA translate into the
  sequence of a protein?
• What is the genetic code?
• How do you translate the "four-letter code" of
  mRNA into the "20-letter code" of proteins?
• And what are the mechanics like? There is no
  obvious chemical affinity between the purine and
  pyrimidine bases and the amino acids that make
  protein.
• As a "way out" of this dilemma, Crick proposed
  "adapter molecules" - they are tRNAs!

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The Collinearity of Gene and Protein Structures

• Watson and Crick's structure for DNA, together
  with Sanger's demonstration that protein
  sequences were unique and specific, made it seem
  likely that DNA sequence specified protein
  sequence
• Yanofsky provided better evidence in 1964 he
  showed that the relative distances between
  mutations in DNA were proportional to the
  distances between amino acid substitutions in E.
  coli tryptophan synthase

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Elucidating the Genetic Code

• How does DNA code for 20 different amino acids?
• 2 letter code would allow for only 16 possible
  combinations.
• 4 letter code would allow for 256 possible
  combinations.
• 3 letter code would allow for 64 different
  combinations
• Is the code overlapping?
• Is the code punctuated?

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The Nature of the Genetic Code

• A group of three bases codes for one amino acid
• The code is not overlapping
• The base sequence is read from a fixed starting
  point, with no punctuation
• The code is degenerate (in most cases, each amino
  acid can be designated by any of several
  triplets)

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How the code was broken

• Assignment of "codons" to their respective amino
  acids was achieved by in vitro biochemistry
• Marshall Nirenberg and Heinrich Matthaei showed
  that poly-U produced polyphenylalanine in a
  cell-free solution from E. coli
• Poly-A gave polylysine
• Poly-C gave polyproline
• Poly-G gave polyglycine
• But what of others?

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Getting at the Rest of the Code

• Work with nucleotide copolymers (poly (A,C),
  etc.), revealed some of the codes
• But Marshall Nirenberg and Philip Leder cracked
  the entire code in 1964
• They showed that trinucleotides bound to
  ribosomes could direct the binding of specific
  aminoacyl-tRNAs
• By using C-14 labelled amino acids with all the
  possible trinucleotide codes, they elucidated all
  64 correspondences in the code

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Features of the Genetic Code

• All the codons have meaning 61 specify amino
  acids, and the other 3 are "nonsense" or "stop"
  codons
• The code is unambiguous - only one amino acid is
  indicated by each of the 61 codons
• The code is degenerate - except for Trp and Met,
  each amino acid is coded by two or more codons
• First 2 codons of triplet are often enough to
  specify amino acid. Third position differs
• Codons representing the same or similar amino
  acids are similar in sequence (Glu and Asp)

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tRNAs

• tRNAs are interpreters of the genetic code
• Length 73 95 bases
• Have extensive 2o structure
• Acceptor arm position where amino acid attached
• Anticodon complementary to mRNA
• Several covalently modified bases
• Gray bases are conserved between tRNAs

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tRNAs 2o vs 3o Structure
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Third-Base Degeneracy

• Codon-anticodon pairing is the crucial feature of
  the "reading of the code"
• But what accounts for "degeneracy" are there 61
  different anticodons, or can you get by with
  fewer than 61, due to lack of specificity at the
  third position?
• Crick's Wobble Hypothesis argues for the second
  possibility - the first base of the anticodon
  (which matches the 3rd base of the codon) is
  referred to as the "wobble position"

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The Wobble Hypothesis

• The first two bases of the codon make normal
  H-bond pairs with the 2nd and 3rd bases of the
  anticodon
• At the remaining position, less stringent rules
  apply and non-canonical pairing may occur
• The rules first base U can recognize A or G,
  first base G can recognize U or C, and first base
  I can recognize U, C or A (I comes from
  deamination of A)
• Advantage of wobble dissociation of tRNA from
  mRNA is faster and protein synthesis too

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AA Activation for Prot. Synth.

• Codons are recognized by aminoacyl-tRNAs
• Base pairing must allow the tRNA to bring its
  particular amino acid to the ribosome
• But aminoacyl-tRNAs do something else activate
  the amino acid for transfer to peptide
• Aminoacyl-tRNA synthetases do the critical job -
  linking the right amino acid with "cognate" tRNA
• Two levels of specificity - one in forming the
  aminoacyl adenylate and one in linking to tRNA

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Aminoacyl-tRNA Synthetase

• Amino acid tRNA ATP ? aminoacyl-tRNA AMP
  PPi
• Most species have at least 20 different
  aminoacyl-tRNA synthetases.
• Typically one enzyme is able to recognize
  multiple anticodons coding for a single amino
  acids (I.e serine 6 different anticodons and only
  one synthetase)
• Two step process
• Activation of amino acid to aminoacyladenylate
• Formation of amino-acyl-tRNA

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Aminoacyladenylate Formation
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Aminoacyl-tRNA Synthetase Rxn
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Specificity of Aminoacyl-tRNA Synthetases

• Anticodon and structure features of acceptor arm
  of specific tRNAs are important in enzyme
  recognition
• Synthetases are highly specific for substrates,
  but Ile-tRNA synthetase has 1 error rate.
  Sometimes incorporates Val.
• Ile-tRNA has proof reading function. Has
  deacylase activity that "edits" and hydrolyzes
  misacylated aminoacyl-tRNAs

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Translation

• Slow rate of synthesis (18 amino acids per
  second)
• In bacteria translation and transcription are
  coupled. As soon as 5 end of mRNA is synthesized
  translation begins.
• Situation in eukaryotes differs since
  transcription and translation occur in different
  cellular compartments.

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Ribosomes

• Protein biosynthetic machinery
• Made of 2 subunits (bacterial 30S and 50S,
  Eukaryotes 40S and 60S)
• Intact ribosome referred to as 70S ribosome in
  Prokaryotes and 80S ribosome in Eukaryotes
• In bacteria, 20,000 ribosomes per cell, 20 of
  cell's mass.
• Mass of ribosomes is roughly 2/3 RNA

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Prokaryotic Ribosome Structure

• E. coli ribosome is 25 nm diameter, 2520 kD in
  mass, and consists of two unequal subunits that
  dissociate at lt 1mM Mg2
• 30S subunit is 930 kD with 21 proteins and a 16S
  rRNA
• 50S subunit is 1590 kD with 31 proteins and two
  rRNAs 23S rRNA and 5S rRNA

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Eukaryotic Ribosome Structure

• Mitochondrial and chloroplast ribosomes are quite
  similar to prokaryotic ribosomes, reflecting
  their supposed prokaryotic origin
• Cytoplasmic ribosomes are larger and more
  complex, but many of the structural and
  functional properties are similar
• 40S subunit contains 30 proteins and 18S RNA.
• 60S subunit contains 40 proteins and 3 rRNAs.

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Ribosome Assembly

• Assembly is coupled w/ transcription and pre-rRNA
  processing

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Ribosome Structure

• Crystal structure of ribosome is known
• mRNA is associated with the 30S subunit
• Two tRNA binding sites (P and A sites) are
  located in the cavity formed by the association
  of the 2 subunits.
• The growing peptide chain threads through a
  tunnel that passes through the 40S (30S in
  bacteria) subunit.

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Mechanics of Protein Synthesis

• All protein synthesis involves three phases
  initiation, elongation, termination
• Initiation involves binding of mRNA and initiator
  aminoacyl-tRNA to small subunit, followed by
  binding of large subunit
• Elongation synthesis of all peptide bonds - with
  tRNAs bound to acceptor (A) and peptidyl (P)
  sites.
• Termination occurs when "stop codon" reached

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Identification of Initiator Codon in Prokaryotes

• Involves binding of initiator tRNA
  (N-formylmethionyl-tRNA) to initiator codon
  (first AUG)
• The 30S subunit scans the mRNA for a specific
  sequence (Shine-Dalgarno Sequence) which is just
  upstream of the initiator codon. 16S RNA is
  involved in recognition of S-D sequence.

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Prokaryotic Translational Initiation

• Formation of Initiation complex involves protein
  initiation factors
• IF-3 keeps ribosome subunits apart
• IF-2 identifies and binds initiator tRNA. IF-2
  must bind GTP to bind tRNA.
• IF-1, IF-2, and IF-3 bind to 30S subunit to form
  initiation complex
• Once 50S subunit binds initiation complex, GTP is
  hydrolyzed, initiator tRNA enters P-site and IFs
  disassociate

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Eukaryotic Initiation of Translation

• No S-D sequence.
• CAP binding protein (CBP) 5 end of mRNA by
  binding to 5 CAP structure
• An initiation complex forms with CBP, initiation
  factors and the 40S subunit.
• The complex then scans the mRNA looking for the
  first AUG closest to the 5 end of the mRNA
• eIF-2 analogous to IF-2, transfers tRNA to P
  sight. GTP hydrolysis involed in release

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Chain Elongation

• Three step process
• Position correct aminoacyl-tRNA at acceptor site
• Formation of peptide bond between peptidyl-tRNA
  at P site with aminoacyl-tRNA at A site.
• Shifting mRNA by one codon relative to ribosome.

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• Elongation Factor Tu (EF-Tu) binds to
  aminoacyl-tRNA and delivers it to the A site of
  the ribosome
• When EF-Tu binds GTP a conformational change
  occurs allowing it to bind to aminoacyl-tRNA.

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• EF-Tu-tRNA complex enters the ribosome and
  positions new tRNA at A site.
• If the anticodon matches the codon, GTP is
  hydrolyzed and EF-Tu releases the tRNA and then
  exits the ribosome.

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Recycling of EF-Tu

• After leaving the ribosome EF-Tu-GDP complex
  associates with EF-Tscausing GDP to disassociate.
• When GTP bind to the EF-Tu/EF-Ts complex, EF-Ts
  disassociates and EF-Tu can bind another tRNA

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Peptide Bond formation
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Formation of Peptide Bond

• Once the peptide bond forms, the mRNA band shifts
  to move the new peptidyl-tRNA into the P-site and
  moves the deaminacyl-tRNA from the E-site
• Binding of EF-GTP to ribosome promotes the
  translocation
• Hydrolysis of EF-GTP to EF-GDP is required to
  release EF from ribosome and new cycle of
  elongation could occur

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More on elongation

• Growing peptide chain then extends into the
  tunnel of the 50S subunit.
• Floding of the native protein does not occur
  until the peptide exits the tunnel
• Folding is facilitated by chaperones that are
  associated with the ribosome
• To ensure the correct tRNA enters the A site, the
  16S RNA is involved in determing correct
  codon/anticodon pairing at positions 1 and 2 of
  the codon.

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Eukaryotic elongation process

• Similar to what occurs in prokaryotes.
• Analogous elongation factors.
• EF-1a EF-Tu ? docks tRNA in A-site
• EF-1b EF-Ts ? recycles EF-Tu
• EF-2 EF-G ? involved in translocation process

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Peptide Chain Termination

• Proteins known as "release factors" recognize the
  stop codon (UGA, UAG, or UAA) at the A site
• In E. coli RF-1 recognizes UAA and UAG, RF-2
  recognizes UAA and UGA.
• RF-3 binds GTP and enhances activities of RF-1
  and 2.
• Presence of release factors with a nonsense codon
  at A site transforms the peptidyl transferase
  into a hydrolase, which cleaves the peptidyl
  chain from the tRNA carrier
• Hydrolysis of GTP is required for disassociation
  of RFs, ribosome subunit and new peptide

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Protein Synthesis is Expensive!

• For each amino acid added to a polypeptide chain,
  1 ATP and 3 GTPs are hydrolyzed.
• This is the release of more energy than is needed
  to form a peptide bond.
• Most of the energy is need to over-come entropy
  losses

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Regulation of Gene Expression
RNA Processing
mRNA
RNA Degradation
AAAAAA
5CAP
Active enzyme
Post-translational modification
Protein Degradation
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Regulation of Protein Synthesis

• Regulation could occur at two levels in
  translation
• Initiation formation of the initiation complex
• Elongation elongation could be stalled by if an
  mRNA contains rare codons

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Regulation of Globin gene translation by heme

• When heme is low, HCI kinase phosphorylates
  eIF-2-GDP complex,
• GEF binds tightly to phosphorylated eiF-2-GDP
  complex
• prevents recycling of eIF-2-GDP and stops
  translation

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Regulation of the trp operon

• Transcription and translation are tightly coupled
  in E. coli.
• When Trp is aundant, transcription of the trp
  operon is repressed.
• The mechanism of this repression is related to
  translation of the

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