Protein synthesis

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Protein synthesis The three roles of RNA in
translation mRNA genetic information tRNA
the key to deciphering the code. rRNA
attracting the mRNA and catalyzing peptide-bond
formation. mRNA carries information from DNA in
a three-letter genetic code Triplet code (4364
codes for 20 amino acids) Start codon
AUG(specifies methionine in all proteins in pro.
and eukarytoes) Stop codons UAA, UGA, and
UAG Structure of tRNA Anticodon loop D
loop T?CG loop
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Wobble pairing G?C or U U?A or G I?U, C, or
A Aminoacyl-tRNA synthetases 30 to 40 diffenent
tRNAs in bacteria and About 50 in animal and
plants cells. 20 different aminoacyl-tRNA
synthetases Ribosomes are protein-synthesizing
machines The steps in protein synthesis
Initiation---Elongation---Termination
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AUG is the initiation signal in mRNA 16S rRNA in
bacteria 5' cap Kozak sequence is mRNA
5'-ACCAUGG Ribosomes provide three tRNA-binding
sites during protein elongation A site (for
aminoacyl-tRNA) P site (for peptidyl-tRNA) Prote
in synthesis inhibitors Antibiotics Puromycine St
reptomycin Chloramphenicol Tetracycline Diphtheria
toxin
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• Protein synthesis
• ? The three roles of RNA in translation
• mRNA genetic information
• tRNA the key to deciphering the code.
• rRNA attracting the mRNA and catalyzing
  peptide-bond formation.
• ? mRNA carries information from DNA in a
  three-letter genetic code
• Triplet code (4364 codes for 20 amino acids)
• Start codon AUG(specifies methionine in all
  proteins in pro. and eukarytoes)
• Stop codons UAA, UGA, and UAG
• ? Experiments with synthetic mRNAs and
  trinucleotides break the genetic code
• When synthetic mRNAs were used to direct in vitro
  protein synthesis, polypeptides formed much more
  inefficiently than when natural mRNAs were used,
  and the lengths of the newly made polypeptide
  chains were variable. With the use of real mRNAs,
  it was soon discovered that AUG encoded
  methionine at the start of almost all proteins
  and that three trinucleotides UAA, UGA, and UAG
  that did not encode any amino acid were stop
  codons

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• ? Folded structure of tRNA is intergral to its
  function
• All tRNAs have two functions to chemically link
  to a particular amino acid and to recognize a
  codon in mRNA so that the amino acid can be added
  to a growing peptide chain
• Each tRNA molecule is recognized by one and only
  one of the 20 enzymes called aminoacyl-tRNA
  synthetases
• Once its correct amino acid is attached, a tRNA
  then recognizes a codon in mRNA
• 30 to 40 diffenent tRNAs in bacteria and about 50
  in animal and plants cells
• There is not a unique tRNA for every single codon
• Four stems are short double helices stabilized by
  base pairing
• Three of the four stems have loops cintaining
  seven or eight vases at their ends
• Anticodon loop
• D loop
• T?CG loop UUCG The first uridylate is
  methylated to become a thymidylate the second is
  rearranged into a pseudouridylate, in which the
  ribose is attached to carbon 5 instead of to
  nitrogen 1
• acceptor arm an amino aicd can be attached to
  the unlooped amino acid acceptor stem.

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• Wobble pairing the capability of a single tRNA
  anticodon to recognize more than one, but not
  every, codon corresponding to a given amino acid.
  This broader recongnition can occur because of
  so-called wobble pairing between the third base
  in a codon and the first base in the
  corresponding anticodon
• The G-U base pair, which structurally fits almost
  as well as the standard G-C pair. Thus a given
  anticodon with G in the first (wobble) position
  can base-pair with the two corresponding codons
  that have either pyrimidine (C or U) in the third
  position (UUU and UUC of mRNA - GAA of anticodon)
• One of the most unusual wobble-position bases in
  plants and animals is inosine, a deaminated
  product of adenine, which can base-pair with A,
  C, and U. A given tRNA with inosine in the wobble
  position thus can recognize the corresponding
  mRNA triplets with A, C, or U in the third codon
  position. For this reason, inosine-containing
  tRNAs are heavily employed in translation fo the
  synonymous codons that specify a single amino
  acid
• ? Aminoacyl-tRNA synthetase activate tRNA
• The 20 different aminoacyl-tRNA synthetases, each
  or which recognizes one amino acid and all its
  compatible, or cognate, tRNAs
• ARS link an amino acid to the free 2' or 3'
  hydroxyl of the ribose of the adenosine at the
  3'-terminus of tRNA
• This two-step linkage reaction requires the
  cleavage of an ATP molecules

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• In the first step, enzyme(aminoacyl-AMP), In the
  second step, aminoacyl-tRNA
• About half of the ARSs transfer the aminoacyl
  group to the 2' hydroxyl of the terminal
  adenosine (class I), and about half, to the 3'
  hydroxyl (class II)
• The resulting aminoacyl-tRNA retains the energy
  of the ATP, and the amino acid residue is said to
  be activated
• ? Each tRNA molecule is recognized by a specific
  aminoacyl-tRNA synthetase
• How each ARS identifies cognate tRNAs is not yet
  understood
• One ARS can add the same amino acid to two ( or
  more) different tRNAs with different anticodons
  encoding the same amino acid
• Therefore each of these tRNAs must have a similar
  binding site that is recognized by the synthetase
• Perhaps the most logical identity site in a tRNA
  molecule is the anticodon itself
• ? Ribosomes are protein-synthesizing machines
• mRNA and aminoacyl-tRNA are brought together by
  their mutual binding to the most abundant
  RNA-protein complex in the cell-the ribosome
• This two-part machine directs the elongation of a
  polypeptide at a rate of three to five amino
  acids added per second
• Structure of large subunits and small subunits of
  ribosome in prokaryotes and eukaryotes

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• ? The steps in protein synthesis
• Initiation---Elongation---Termination.
• ? AUG is the initiation signal in mRNA
• There are at least two types of tRNAiMet that
  can initiate protein synthesis, and another that
  can incorporaate methionine within the growing
  protein chain
• The same enzyme, methionyl-tRNA synthetase, can
  attach methionine to both tRNAs, but only
  methionyl-tRNAiMet can bind to the small
  ribosomal subunit to begin the process of protein
  synthesis
• In bacteria, N-formylmethionyl-tRNAfMet
• In most bacteria, the small ribosomal subunit
  identifies initiation sites through the
  interaction of short nucleotide sequences in the
  small 16S rRNA and the mRNA
• On the mRNA this Shine-Dalgarno sequence is near
  a prtoen start site and complementary to a
  sequence at or very near the 3' end of the
  16S-rRNA molecule
• Thus bacterial rRNA plays a direct role in
  recruiting a ribosome to a protein start site on
  the mRNA
• In eukaryotic cells the mechanism by which a
  small ribosomal subunit finds start sites is not
  fully understood

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• The first signal thought to be recognized is the
  5' cap present on all eukaryotic mRNAs however,
  some viral mRNAs, which are translated by the
  host-cell machinery in ingected eukaryotic cells,
  lack a 5'cap. In this case recognition occurs
  with the aid of additional protein factors
• Usually, after cap recognition, the bound
  ribosomal subunit then is thought to slide along
  the mRNA to locate an AUG
• Frequently the first AUG is used, but the
  presence of certain nucleotides surrounding the
  initiating AUG greatly increases the
  effectiveness of initiation. This sequence,
  reffered to an a Kozak swquence is mRNA
  5'-ACCAUGG-
• mRNAs without the 5' cap are translated very
  poorly, and mutations introduced into Kozak
  sequences greatly decrease initiation frequency
• It is generally agreed that initiation of
  translation of most eukaryotic mRNAs involves
  recognition of the cap followed either by use of
  the first downstream AUG or by the locating of a
  5'-proximal AUG with a consensus sequence
  surrounding the AUG codon

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• ? Initiation factors, tRNA, mRNA, and the small
  ribosomal subunit form an initiation complex
• Initiation factors help the small ribosomal
  subunit find the initiation site
• Without these proteins, the complex of mRNA,
  Met-tRNAiMet, and the small ribosomal subunit
  poised at the AUG initiation codon does not form
• In prokaryotes, IF3 is critical in finding the
  AUG. In eukaryotes, the large eIF4 complex helps
  to ensure that the 5' end of the mRNA is
  single-stranded (the factor server both tobind
  the cap and to unwind any secondary structure
  that may exist) it also ensures that the 5' end
  is ready for the small subunit to locate the AUG
• GTP hydrolysis provides the energy required for
  many of the steps in protein synthesis. For
  example, the positioning of the large subunit in
  the bacterial initiation complex is aided by IF2.
  GTP and requires GTP hydrolysis
• ? Ribosomes provide three tRNA-binding sites
  during protein elongation
• A site (for aminoacyl-tRNA)
• P site (for peptidyl-tRNA)
• E site (transiently occupied by the deacylated
  end of the tRNA)
• Met-tRNAiMet enters the P position

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• The incoming aminoacyl-tRNA is then bound to the
  A site
• The hydrolysis furnishes the energy for this
  translocation of the peptidyl-tRNA
• In bacterial protein synthesis the 23S rRNA in
  the large ribosomal subunit itself may carry out
  the crucial peptidyltransferase function
• The actual peptide-synthesis reaction involves
  23S rRNA
• Clearly, many of the reactions neccessary for
  peptide synthesis are RNA mediated
• ? Polypeptide termination requires protein
  factors that specifically recognize UAA, UAG and
  UGA
• The stop codons signal the release of the
  peptidyl-tRNA complex when recognized by protein
  termination factors
• There are at least two such factors in
  prokaryotes and probably also in eukaryotes
• Almost simultaneously the complex divides into an
  uncharged tRNA molecule lacking an attached amino
  acid and a newly completed protein chain

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• ? Protein synthesis inhibitors Antibiotics
• Antibiotics are bacterial or fungally produced
  substances that inhibit the growth of other
  organisms
• Puromycin is an aminoacyl-tRNA analog (for
  tyrosyl-tRNA)
• This substance, which resembles the 3' end
  of Tyr-tRNA, causes the premature termination of
  polypeptide chain synthesis
• Streptomycin
• Chloramphenicol
• Tetracycline
• Diphtheria toxin