Biochemistry

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Biochemistry

• Chen Yonggang
• Zhejiang University Schools of Medicine

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Translation, making protein following nucleic
acid directions
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Bodega Bay, Sonoma County
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Breakfast at The Tides, Bodega Bay
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The process of using base pairing language to
create a protein is termed Translation

• Any process requires
• A mechanism Ribosome
• Information-directions mRNA
• Raw materials amino acids / tRNA
• Energy ATP
• Any process has stages
• Beginning Initiation
• Middle Elongation
• End Termination

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Translation requires a Dictionary

• The dictionary of Translation is called the
  Genetic Code Table 6.1
• Correlates mRNA with Protein
• 3 nucleotides 1 amino acid 43 64
• 4 possible nts 20 possible aa
• 3 nucleotides read 5?3 are called a codon
• Codes for 1 amino acid

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The Genetic Code
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The Genetic Code

• Triplet made of codons
• Non-overlapping read sequentially
• Unpunctuated once started, set frame
• Degenerate gt than one codon/AA
• Nearly universal mitochondrial code
• Start signals AUGmet
• Stop signals UAG, UAA, UGA

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Players in Translation

• Ribosome the machinery
• mRNA the information
• Aminoacyl-tRNA the translator!
• Amino Acids/tRNA
• ATP

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Ribosomes are ribonucleoprotein complexes table
6.7
PROCARYOTIC
EUCARYOTIC
80 S 40S 60S RNA 5S, 5.8S,18S,28S PROTEINS 84
70 S 30S 50S RNA 5S, 16S, 23S PROTEINS 55
Small subunit
Large Subunit
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Ribosomes must be assembled with an mRNA

• The initiation process requires protein factors
• A mRNA must be recognized and reading frame must
  be set
• Aminoacyl-tRNAs must be available

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5
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Since the Translator is the Aminoacyl-tRNA, it
must be important

• Cells have 30 tRNAs
• tRNAs are redundant for some amino acids
• Cells have 20 Aminoacyl-tRNA Synthetases
• Aminoacyl-tRNA synthetases recognize 1 amino acid
  and 1 or more tRNAs
• Aminoacylation is very precise

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Aminoacyl-tRNA Synthetases are critical to
Translation

• 1 Aminoacyl-tRNA Synthetase recognizes 1 Amino
  Acid and binds it
• 1 Aminoacyl-tRNA Synthetase recognizes 1 or more
  tRNAs specific for 1 amino acid
• The aminoacyl-tRNA Synthetase catalyzes a two
  step reaction which overall is
• AAx tRNAx ATP AAx-tRNAx AMP PPi
• Page 239

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The first step involves forming an enzyme-bound
aminoacyl adenylate
The hydrolysis of the PPi makes the process
irriversible
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The second step transfers the amino acid to the
3OH of the tRNA, retaining the energy of the
adenylate
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tRNAs fold into L-shaped structuresFigure 2.59
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Functional Sites of tRNAsFigure 2.58

• CCAOH 3 Acceptor Sequence
• Amino acid acceptor stem
• D stem and loop
• Extra loop
• Anticodon stem and loop
• Anticodon
• TyC stem and loop
• 5 Terminus

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The anticodon forms antiparallel base pairs with
a codon in the mRNA

• Each tRNA has a unique anticodon
• There are 61 codons which base pair with tRNA
  anticodons, most pairing is Watson-Crick but
  Wobble in the 5 base of the anticodon allows
  degeneracy
• 3 codons do not normally base pair with
  anticodons-UAA, UAG, UGA. The lack of a
  complementary anticodon-Termination Codons

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Wobble allows one codon to base pair with up to
three anticodons
Base stacking in the anticodon assures that bases
2 and 3 of the anticodon will follow Watson-Crick
rules. Base 1 can wobble
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Depending on base 1 it can pair with 1,2 or 3
bases

• If the wobble base is U, it can H bond to A
  (expected) or G (unexpected).
• If the wobble base is G, it can H bond to C
  (expected) or U (unexpected).
• A and C form only the expected base pairs.
• Inosine in the wobble position can H bond to A,
  C, and U.

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Thus 31 tRNAs can read 61 codons
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Translation takes place in three stages

• Initiation-- once per protein it gets the system
  in motion
• Elongation-- repeated for each codon in the mRNA
  making a peptide bond
• Termination-- finishes and releases the newly
  synthesized protein

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Initiation

• A common mechanism

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Procaryotic initiation assembles the
pre-translational complex

• Mechanism is similar for eucaryotes and
  procaryotes differences are important
• Components
• Small subunit containing a specific mRNA
  sequence(Shine-Dalgarno) which guides the mRNA
  into correct position for reading frame relative
  to the 16S rRNA
• Proteinaceous initiation factors
• Initiator AA-tRNA
• mRNA(monocistronic for eucaryotes, polycistronic
  for procaryotes)

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Differences in the process provide the basis for
specific antibiotic action

• Procaryotes
• 30S ribosomal subunit
• IF-1, IF-2, IF-3
• fMet-tRNAMetF
• GTP

• Eucaryotes
• 40S ribosomal subunit
• eIF-2a, eIF-3, eIF-4a, eIF-4c, eIF-4e, eIF-4g,
  eIF-5, eIF-6
• Met-tRNAMeti
• GTP

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Initiation Factors have Specific Roles

• Procaryotes
• IF-3 binds 30S
• IF-2 binds initiator AA-tRNA
• IF-1 GTP hydrolysis
• RNARNA base pairing indexes mRNA

• Eucaryotes
• eIF-2 itRNA Binding
• eIF-3 40S anti-association
• eIF-4g binds mRNA
• eIF-4e cap binding
• eIF-4a mRNA indexing
• eIF-4c ribosomal i AA-tRNA
• eIF-5 GTP hydrolysis
• eIF-6 60S anti-association

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In procaryotes IFs 1,2 and 3 are needed to begin
IF-3 is an 30S anti-association factor IF-2 binds
and preps initiator AA-tRNA IF-1 is a GTP binding
hydrolase These allow the association of the 30S,
Met-tRNA metF and factors to bind in preparation
for mRNA and 50S binding
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(No Transcript)
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Initiation is similar for pro- and
eucaryotesDevlin 6.7
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Intiation occurs once per translational cycle

• The preinitiation complex is formed on the small
  subunit
• GTP is bound to initiation factors. GTP
  hydrolysis carries out a process and drives a
  conformational change which leads to the next
  activity
• The mRNA is indexed to appropriate AUG codon
• The mRNA is locked into the cleft between small
  and large subunits
• Addition of the large subunit creates A , P and E
  sites on the ribosome
• The initiator AA-tRNA is locked into the P site

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Eucaryotic initiation is similar
Devlin 6.7
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Eucaryotic initiation has differences

• The mRNA is not indexed by the ribosomal rRNA
  (eukaryotic mRNAs do not have Shine-Dalgarno
  sequence)
• Cap binding is essential for initiation
• The initiation complex does not use formylated
  methionine but does use a specific initiator
  Methionine-specific aminoacyl-tRNA for initiation
• Protein synthesis occurs at the first AUG

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The association of all initiation components
creates a 70S ribosome with initiator tRNA in the
P site
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Elongation

• A repeated experience

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Once initiation is complete the ribosome is ready
for elongation

• Elongation is the process of addition of amino
  acids to the C-terminus of the growing
  polypeptide
• Synthesis of each peptide bond requires energy
  derived from the cleavage of the AA-tRNA ester
  bond. The ribosomal enzyme doing this is called
  Peptidyl Transferase
• Elongation is repeated as many times as there are
  codons in the mRNA

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As is the case for initiator tRNA all
aminoacyl-RNAs must be present for protein
synthesis

• Good nutrition requires that all amino acids must
  be available in the diet
• For procaryotes most can be synthesized at an
  expense of energy
• Eucaryotes are able to form some but not all
  amino acids, thus some are essential in the diet

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Pools of AA-tRNAs are formed by the
Aminoacyl-tRNA Synthetases

• AA-tRNA synthetases recognize 2o and 3o
  structure near the TyC,D, and extra loop and the
  acceptor stem on the L-shaped tRNA molecules
• AA-tRNA synthetases recognize 3-dimensional
  structure and functional groups of the amino
  acids
• As we saw earlier, AA-tRNA synthetases use ATP to
  form a high-energy ester bond at the 3OH on the
  tRNA

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Once an AAx-tRNAx is formed, the Amino Acid
becomes Invisible

• The ribosome mediates the association between
  codons on the mRNA and anticodons on the tRNA
• Specificity of AA incorporation depends upon the
  anticodon of the tRNA
• Whatever is on the tRNA will be incorporated into
  the protein at the site
• The tRNA adapts the AA to the specified site

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Following Initiation the Ribosome has 3
functional sites

• A site-aminoacyl-tRNA binding site incoming
  AA-tRNA, only initiator AA-tRNA goes to the P
  site
• P site-peptidyl-tRNA binding siteattachment of
  growing polypeptide site
• E site-spent tRNA exit site

A
P
E
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Each elongation cycle requires elongation factors

• Procaryotes
• EF-T AA-tRNA binding to A site, GTP
  binding/hydrolysis
• EF-G GTP hydrolysis, ribosomal conformational
  change, index peptidyl-tRNA to P site, expulsion
  of spent tRNA from E site

• Eucaryotes
• EF-1 AA-tRNA binding to A site, GTP
  binding/hydrolysis
• EF-2 GTP hydrolysis, ribosomal conformational
  change, index peptidyl-tRNA to P site, expulsion
  of tRNA from E site

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In procaryotes, under the control of EF-T, a
second aminoacyl-tRNA is bound in the A site
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In eucaryotes similar events occur
Devlin 6.8
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Hydrolysis of bound GTP changes the conformation
of the Ribosome

• The conformational change locks the
  aminoacyl-tRNA into the A site
• Brings the anticodon in close approximation with
  the codon
• Prepares the ribosome for binding of another GTP
  binding hydrolase EF-G

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The energy for peptide bond formation derives
from the aminoacyl-tRNA ester bond

• Cleaving the ester bond provides energy for the
  formation of a peptide bond
• Catalysis is most likely provided by an integral
  50/60S ribozyme, the peptidyl transferase, an
  RNA-containing enzyme(parts of the 23s rRNA) in
  the ribosome
• Upon synthesis of the peptide bond, the growing
  polypeptide chain is linked to the tRNA on the P
  site

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Peptidyl transferase synthesizes a peptide bond
forming a dipeptide
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The peptide bond is formed using the energy
derived from the aminoacyl ester bond and moves
the peptide to the A site-bound Aminoacyl-tRNA
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Following peptide bond formation a new factor
drives translocation of the peptide

• Specificity provided by antiparallel
  codon-anticodon pairing between A site-bound
  AA-tRNA and mRNA
• Translocation driven by EF-G/2 catalyzed GTP
  hydrolysis-derived conformational change
• mRNA ratchets 5?3 through the ribosome moving
  the C(codon)AC(anticodon) from A to P site by
  the action of a translocase
• Time to find AA-tRNA is important to fidelity

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EF-G mediated GTP hydrolysis translocates the
mRNA and peptidyl-tRNA expelling the spent tRNA
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Eucaryotic translocation is similar
Devlin 6.8
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This elongation cycle is repeated as many times
as there are codons
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EF-T/1 mediated binding is followed peptide bond
formation and EF-G/2 mediated peptidyl transfer
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Eucaryotic elongation is similar to the
procaryotic process
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Repeat of 3 steps in elongation cycle

• 1. Binding of an incoming AA-tRNA
• 2. Peptide bond formation, catalyzed by
• peptidyl transferase
• 3. translocation, done by translocase

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The growing polypeptide chain remains attached to
the last tRNA added The next codon is UAG
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When a termination codon occupies the the A site
no AA-tRNA will bind

• Termination codons work because no tRNA has a
  complementary anticodon
• When the site is occupied by UAA, UAG or UGA time
  passes without A site occupancy by an AA-tRNA
• This allows binding of release or termination
  factors, proteinssize and shape of tRNAs that
  change the activity of peptidyl transferase to a
  peptidyl hydrolase and thus mediate release of
  the polypeptide from the ribosome

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Termination requires proteinaceous termination
factors

• Procaryotes
• Release Factor GTP binding, GTP hydrolysis,
  conformational change, cleavage of 3-peptidyl-
  CCAOH ester linkage, expulsion of polypeptide,
  dissociation of 30S and 50S subunits

• Eucaryotes
• eRF GTP binding, GTP hydrolysis, conformational
  change, cleavage of 3-peptidyl-CCAOH ester
  linkage, expulsion of polypeptide, dissociation
  of 40S and 60S subunits

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Devlin 6.10
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Polysome

• In both prokaryotes and eukaryotes,
  mRNAs are read simultaneously by numerous
  ribosomes, An mRNA with several ribosomes bound
  to it is referred to as a polysome.

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Posttranslational modification

• Some newly made proteins, both prokaryotic and
  eukaryotic, do not attain their final
  biologically active conformation until they have
  been altered by one or more processing reactions
  called posttranslational modification

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Different ways of modification

• Amino-Terminal and Carboxyl-Terminal
  Modification
• Loss of Signal Sequence the 15 to 30 residues at
  the amino-terminal end of some proteins play a
  role in directing the protein to its ultimate
  destination in the cell. Such signal sequences
  are ultimately removed by peptidase
• Modification of Individual Amino Acids
• The hydroxyl groups of Ser, Thr, and Tyr can
  be phosphorylated , some others can be
  carboxylated and methylated.

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Different ways of modification

• Attachment of Carbohydrate Side Chains such as
  glycoproteins, N-linked oligosaccharides (e.g.
  Asn), O-linked-oligosaccharides(e.g. Ser or Thr)
• Addition of Isoprenyl Groups
• Addition of Prosthetic GroupsTwo examples are
  the biotin molecule of acetyl-CoA carboxylase
  and the heme group of hemoglobin or cytochrome
  c.

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Different ways of modification

• Proteolytic Processing proinsulin and proteases
  such as chymotrypsinogen and trypsinogen(zymogen
  activation)
• Formation of Disulfide Cross-link intrachain or
  interchain disulfide bridges between Cys residues

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Because of differences in translation bacterial
growth can be inhibited by antibiotics
Devlin 6.8
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Eucaryotes can be targeted by microorganisms

• Diphtheria toxin carries out its effects by
  mediating a covalent modification of eEF-2
• NAD EF-2 ADP-Ribose-EF2
  Nicotinamide
• ADP-ribosylated eEF-2 is ineffective, thus
  interrupting polypeptide synthesis

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Whats Next?

• Once made can proteins be modified?
• How is protein folding effected?
• How are proteins exported after synthesis?
• How is protein turnover controlled?

I can hardly wait!