Molecular Biology Fourth Edition

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Molecular BiologyFourth Edition

• Chapter 17
• The Mechanism of Translation I Initiation

Chapter 18 The Mechanism of Translation II
Elongation and Termination
Chapter 19 Ribosomes and Transfer RNA
Robert F. Weaver
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18.1 Direction of Polypeptide Synthesis and mRNA
Translation

• Messenger RNAs are read in the 5?3 direction
• This is the same direction in which they are
  synthesized
• Proteins are made in the amino?carboxyl direction
• This means that the amino terminal amino acid is
  added first

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Strategy to Determine Direction of Translation
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18.2 The Genetic Code

• The term genetic code refers to the set of 3-base
  code words (codons) in mRNA that represent the 20
  amino acids in proteins
• Basic questions were answered about translation
  in the process of breaking the genetic code

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Nonoverlapping Codons

• Each base is part of at most one codon in
  nonoverlapping codons
• In an overlapping code, one base may be part of
  two or even three codones

• AUGUUC
• No overlapping AUG UUC
• Overlapping AUG UGU GUU UUC
• If no overlapping, a change of one base in an
  mRNA would change no more than one a.a. in the
  resulting protein.
• If overlapping, up to three adjacent amino acids
  could be changed.

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No-overlapping

• One base change in TMV mRNA never caused more
  than one a.a. mutation

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No Gaps in the Code

• If the code contained untranslated gaps or
  commas, mutations adding or subtracting a base
  from the message might change a few codons
• Would still expect ribosome to be back on track
  after the next such comma
• Mutations might frequently be lethal
• Many cases of mutations should occur just before
  a comma and have little, if any, effect

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Frameshift Mutations

• Frameshift mutations
• Translation starts AUGCAGCCAACG
• Insert an extra base AUXGCAGCCAACG
• Extra base changes not only the codon in which is
  appears, but every codon from that point on
• The reading frame has shifted one base to the
  left
• Code with commas
• Each codon is flanked by one or more untranslated
  bases
• Commas would serve to set off each codon so that
  ribosomes recognize it
• Translation starts AUGZCAGZCCAZACGZ
• Insert an extra base AUXGZCAGZCCAZACGZ
• First codon wrong, all others separated by Z,
  translated normally

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Frameshift Mutation Sequences
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The Triplet Code

• The genetic code is a set of three-base code
  words, or codons
• In mRNA, codons instruct the ribosome to
  incorporate specific amino acids into a
  polypeptide
• Code is nonoverlapping
• Each base is part of only one codon
• Devoid of gaps or commas
• Each base in the coding region of an mRNA is part
  of a codon

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Breaking the Code

• The genetic code was broken
• Using
• Synthetic messengers
• Synthetic trinucleotides
• Then observing
• Polypeptides synthesized
• Aminoacyl-tRNAs bound to ribosomes
• There are 64 codons
• 3 are stop signals
• Remainder code for amino acids
• The genetic code is highly degenerate

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How to test the the triplet code hypothesis

• In 1961, Nirenberg and Matthaei,
• Poly(U) can be translated into poly-Phe
• Synthetic mRNA of defined sequence can shed light
  on the nature of the code

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First, the codons contained odd number of bases.

• PolyUC or UCUCUCUC.
• Odd number of bases------UCU or CUC will code for
  di-peptide
• Even number of bases-----CUCU or UCUC will code
  for single repeat
• Found. code for poly(ser-leu)---So, the codons
  contained an odd number of bases

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Odd bases!
Poly(UC)
Three bases?
Poly(UUC)
Four bases?
Poly(UAUC)
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Four different bases, only twenty amino
acids Triplet 4x4x464 Two-base codon 4x416 not
enough combination Nine-base codon 49262,144
too many
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The Genetic Code
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Unusual Base Pairs Between Codon and Anticodon

• Degeneracy of genetic code is accommodated by
• Isoaccepting species of tRNA bind same amino
  acid, but recognize different codons
• Wobble, the 3rd base of a codon is allowed to
  move slightly from its normal position to form a
  non-Watson-Crick base pair with the anticodon
• Wobble allows same aminoacyl-tRNA to pair with
  more than one codon (to reduce the number of tRNA
  to translate genetic code)

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Wobble Base Pairs

• Compare standard Watson-Crick base pairing with
  wobble base pairs
• Wobble pairs are
• G-U
• I-A (or C, or U)

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Wobble Position
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Almost Universal Code

• Genetic code is NOT strictly universal
• Certain eukaryotic nuclei and mitochondria along
  with at least one bacterium
• Codons cause termination in standard genetic code
  can code for amino acids Trp, Glu
• Mitochondrial genomes and nuclei of at least one
  yeast have sense of codon changed from one amino
  acid to another
• Deviant codes are still closely related to
  standard one from which they evolved
• Genetic code a frozen accident or the product of
  evolution
• Ability to cope with mutations evolution

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Deviations from Universal Genetic Code
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18.3 The Elongation Mechanism

• Elongation takes place in three steps
• EF-Tu with GTP binds aminoacyl-tRNA to the
  ribosomal A site
• Peptidyl transferase forms a peptide bond between
  peptide in P site and newly arrived
  aminoacyl-tRNA in the A site
• Lengthens peptide by one amino acid and shifts
  it to the A site
• EF-G with GTP translocates the growing
  peptidyl-tRNA with its mRNA codon to the P site

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Elongation in Translation
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A Three-Site Model of the Ribosome

• Puromycin
• Resembles an aminoacyl-tRNA
• Can bind to the A site
• Couple with the peptide in the P site
• Release it as peptidyl puromycin
• If peptidyl-tRNA is in the A site, puromycin will
  not bind to ribosome, peptide will not be
  released
• Two sites are defined on the ribosome
• Puromycin-reactive site (P)
• Puromycin unreactive site (A)
• 3rd site (E) for deacylated tRNA bind to E site
  as exits ribosome

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Puromycin Structure and Activity
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Protein Factors and Peptide Bond Formation

• One factor is T, transfer
• It transfers aminoacyl-tRNAs to the ribosome
• Actually 2 different proteins
• Tu, u stands for unstable
• Ts, s stands for stable
• Second factor is G, GTPase activity
• Factors EF-Tu and EF-Ts are involved in the first
  elongation step
• Factor EF-g participates in the third step

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Elongation Step 1

• Binding aminoacyl-tRNA to A site of ribosome
• Ternary complex formed from
• EF-Tu
• Aminoacyl-tRNA
• GTP
• Delivers aminoacyl-tRNA to ribosome A
• site without hydrolysis of GTP
• Next step
• EF-Tu hydrolyzes GTP
• Ribosome-dependent GTPase activity
• EF-Tu-GDP complex dissociates from ribosome
• Addition of aminoacyl-tRNA reconstitutes ternary
  
• complex for another round of translation
• elongation

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Aminoacyl-tRNA Binding to Ribosome A Site
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Proofreading

• Protein synthesis accuracy comes from charging
  tRNAs with correct amino acids
• Proofreading is correcting translation by
  rejecting an incorrect aminoacyl-tRNA before it
  can donate its amino acid
• Protein-synthesizing machinery achieves accuracy
  during elongation in two steps

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Protein-Synthesizing Machinery

• Two steps achieve accuracy
• Gets rid of ternary complexes bearing wrong
  aminoacyl-tRNA before GTP hydrolysis
• If this screen fails, still eliminate incorrect
  aminoacyl-tRNA in the proofreading step before
  wrong amino acid is incorporated into growing
  protein chain
• Steps rely on weakness of incorrect
  codon-anticodon base pairing to ensure
  dissociation occurs more rapidly than either GTP
  hydrolysis or peptide bond formation

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Proofreading Balance

• Balance between speed and accuracy of translation
  is delicate
• If peptide bond formation goes too fast
• Incorrect aminoacyl-tRNAs do not have enough time
  to leave the ribosome
• Incorrect amino acids are incorporated into
  proteins
• If translation goes too slowly
• Proteins are not made fast enough for the
  organism to grow successfully
• Actual error rate, 0.01 per amino acid is a
  good balance between speed and accuracy

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Elongation Step 2

• One the initiation factors and EF-Tu have done
  their jobs, the ribosome has fMet-tRNA in the P
  site and aminoacyl-tRNA in the A site
• Now form the first peptide bond
• No new elongation factors participate in this
  event
• Ribosome contains the enzymatic activity,
  peptidyl transferase, that forms peptide bond

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Assay for Peptidyl Transferase
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Peptide Bond Formation

• The peptidyl transferase resides on the 50S
  ribosomal particle
• Minimum components necessary for activity are 23S
  rRNA and proteins L2 and L3
• 23S rRNA is at the catalytic center of peptidyl
  transferase

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Elongation Step 3

• When peptidyl transferase has worked
• Ribosome has peptidyl-tRNA in the A site
• Deacylated tRNA in the P site
• Translocation, next step, moves mRNA and
  peptidyl-tRNA one codons length through the
  ribosome
• Places peptidyl-tRNA in the P site
• Ejects the deacylated tRNA
• Process requires elongation factor EF-G which
  hydrolyzes GTP after translocation is complete

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Three-Nucleotide Movement

• Each translocation event moves the mRNA on codon
  length, or 3 nt through the ribosome

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Role of GTP and EF-G

• GTP and EF-G are necessary for translocation
• Translocation activity appears to be inherent in
  the ribosome
• This activity can be expressed without EF-G and
  GTP
• GTP hydrolysis
• Precedes translocation
• Significantly accelerate translocation
• New round of elongation occurs if
• EF-G must be released from the ribosome
• Release depends on GTP hydrolysis

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GTPases and Translation

• Some translation factors harness GTP energy to
  catalyze molecular motions
• These factors belong to a large class of G
  proteins
• Activated by GTP
• Have intrinsic GTPase activity activated by an
  external factor (GAP)
• Inactivated when they cleave their own GTP to GDP
• Reactivated by another external factor (guanine
  nucleotide exchange protein) that replaces GDP
  with GTP

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Structures of EF-Tu and EF-G

• Three-dimensional shapes determined by x-ray
  crystallography
• EF-Tu-tRNA-GDPNP ternary complex
• EF-G-GDP binary complex
• As predicted, the shapes are very similar

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18.4 Termination

• Elongation cycle repeats over and over
• Adds amino acids one at a time
• Grows the polypeptide product
• Finally ribosome encounters a stop codon
• Stop codon signals time for last step
• Translation last step is termination

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Termination Codons

• Three codons are the natural stop signals at the
  ends of coding regions in mRNA
• UAG
• UAA
• UGA
• Mutations can create termination codons within an
  mRNA causing premature termination of translation
• Amber mutation creates UAG
• Ochre mutation creates UAA
• Opal mutation creates UGA

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Amber Mutation Effects in a Fused Gene
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Termination Mutations

• Amber mutations are caused by mutagens that give
  rise to missense mutations
• Ochre and opal mutations do not respond to the
  same suppressors as do the amber mutations
• Ochre mutations have their own suppressors
• Opal mutations also have unique suppressors

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Termination Mutations
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Stop Codon Suppression

• Most suppressor tRNAs have altered anticodons
• Recognize stop codons
• Prevent termination by inserting an amino acid
• Allow ribosome to move on to the next codon

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Release Factors

• Prokaryotic translation termination is mediated
  by 3 factors
• RF1 recognizes UAA and UAG
• RF2 recognizes UAA and UGA
• RF3 is a GTP-binding protein facilitating binding
  of RF1 and RF2 to the ribosome
• Eukaryotes has 2 release factors
• eRF1 recognizes all 3 termination codons
• eRF3 is a ribosome-dependent GTPase helping eRF1
  release the finished polypeptide

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Release Factor Assays
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Dealing with Aberrant Termination

• Two kinds of aberrant mRNAs can lead to aberrant
  termination
• Nonsense mutations can occur that cause premature
  termination
• Some mRNAs (non-stop mRNAs) lack termination
  codons
• Synthesis of mRNA was aborted upstream of
  termination codon
• Ribosomes translate through non-stop mRNAs and
  then stall
• Both events cause problems in the cell yielding
  incomplete proteins with adverse effects on the
  cell
• Stalled ribosomes out of action
• Unable to participate in further protein synthesis

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Non-Stop mRNAs

• Prokaryotes deal with non-stop mRNAs by
  tmRNA-mediated ribosome rescue
• Alanyl-tmRNA resembles alanyl-tRNA
• Binds to vacant A site of a ribosome stalled on a
  non-stop mRNA
• Donates its alanine to the stalled polypeptide
• Ribosome shifts to translating an ORF on the
  tmRNA (transfer-messenger RNA)
• Adds another 9 amino acids to the polypeptide
  before terminating
• Extra amino acids target the polypeptide for
  destruction
• Nuclease destroys non-stop mRNA

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Non-Stop mRNAs

• Prokaryotes deal with non-stop mRNAs by
  tmRNA-mediated ribosome rescue
• tmRNA are about 300 nt long
• 5- and 3-ends come together to form a tRNA-like
  domain (TLD) resembling a tRNA

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Eukaryotic Aberrant Termination

• Eukaryotes do not have tmRNA
• Eukaryotic ribosomes stalled at the end of the
  poly(A) tail contain 0 3 nt of poly(A) tail
• This stalled ribosome state is recognized by
  carboxyl-terminal domain of a protein called
  Ski7p
• Ski7p also associates tightly with cytoplasmic
  exosome, cousin of nuclear exosome
• Non-stop mRNA recruit Ski7p-exosome complex to
  the vacant A site
• Ski complex is recruited to the A site
• Exosome, positioned just at the end of non-stop
  mRNA, degrades that RNA
• Aberrant polypeptide is presumably destroyed

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Exosome-Mediated Degradation

• This stalled ribosome state is recognized by
  carboxyl-terminal domain of a protein called
  Ski7p
• Ski7p also associates tightly with cytoplasmic
  exosome, cousin of nuclear exosome
• Non-stop mRNA recruit Ski7p-exosome complex to
  the vacant A site
• Ski complex is recruited to the A site

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Premature Termination

• Eukaryotes deal with premature termination codons
  by 2 mechanisms
• NMD (nonsense-mediated mRNA decay)
• Mammalian cells use a downstream destabilizing
  element
• Yeast cells appear to recognize a premature stop
  codon
• NAS (nonsense-associated altered splicing)
• Senses a stop codon in the middle of a reading
  frame
• Changes the splicing pattern so premature stop
  codon is spliced out of mature mRNA
• Both mechanisms require Upf1

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Mammalian NMD

• NMD in mammalian cells involves a downstream
  destabilizing element
• Upf1
• Upf2
• Bind to mRNA at exon-exon junction that measures
  distance to a stop codon
• Codon far enough upstream
• Looks like a stop codon
• Activates downstream destabilizing element to
  degrade mRNA

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Yeast NMD

• Yeast cells appear to recognize a premature stop
  codon by the absence of a normal 3-UTR or poly
  (A) nearby
• Ribosome stopping at premature stop codon moves
  to an upstream AUG
• This may mark the mRNA for destruction

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NAS and NMD Models
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Use of Stop Codons to Insert Unusual Amino Acids

• Unusual amino acids are incorporated into growing
  polypeptides in response to termination codons
• Selenocysteine uses a special tRNA
• Anticodon for UGA codon
• Charged with serine then converted to
  selenocysteine
• Selenocysteyl-tRNA escorted to ribosome by
  special EF-Tu
• Pyrrolysine uses a special tRNA synthetase that
  joins preformed pyrrolysine with a special tRNA
  having an anticodon recognizing UAG

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18.5 Posttranslation

• Translation events do not end with termination
• Proteins must fold properly
• Ribosomes need to be released from mRNA and
  engage in further translation rounds
• Folding is actually a cotranslational event
  occurring as nascent polypeptide is being made

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Folding Nascent Proteins

• Most newly-made polypeptides do not fold properly
  alone
• Polypeptides require folding help from molecular
  chaperones
• E. coli cells use a trigger factor
• Associates with the large ribosomal subunit
• Catches the nascent polypeptide emerging from
  ribosomal exit tunnel in a hydrophobic basket to
  protect from water
• Archaea and eukaryotes lack trigger factor, use
  freestanding chaperones

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Release of Ribosomes from mRNA

• Ribosomes do not release from mRNA spontaneously
  after termination
• Help is required from ribosome recycling factor
  (RRF) and EF-G
• RRF resembles a tRNA
• Binds to ribosome A site
• Uses a position not normally taken by a tRNA
• Collaborates with EF-G in releasing either 50S
  ribosome subunit or whole ribosome