Ribosomes and Transfer RNA

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Chapter 19

• Ribosomes and Transfer RNA

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Ribosome composition

• Prokaryotes
• 30S
• S1-S21 16S rRNA
• 50S
• L1-L33 (L34 not visible) 5S rRNA and 23S rRNA
• Eukaryotes
• 40S
• About 30 proteins 18S rRNA
• 60S
• About 40 proteins 5S, 5.8S, and 28S rRNA

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Fig. 19.5
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Polysomes -P625

• Both prokaryotes and eukaryotes have polysomes

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Polysomes -P624

• Most mRNA are translated by more than one
  ribosome at a time the result, a structure in
  which many ribosomes translate an mRNA in tandem,
  is called a polysome.
• In eukaryotes, polysomes are found in the
  cytoplasm. In prokaryotes, transcription of a
  gene and translation of the resulting mRNA occur
  simultaneously. Therefore, many polysomes are
  found associated with an active gene.

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Eukaryotes
74 ribosome translating simultaneously
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Prokaryotes
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19.2 Transfer RNA
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The discovery of tRNA

• Zamecnik and et al.,1957
• pH5 enzyme fraction works with ribosomes to
  direct translation of added mRNAs.

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Mix RNA with pH5 enzyme, ATP and
14CLeucine Charging of tRNA with an
amino acid
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Mix 14CLeucine-charged pH 5 RNA with
microsome
Incorporation of leucine from leucyl-tRNA in to
the nascent protein on ribosome
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Summary

• Transfer RNA was discovered as a small RNA
  species independent of ribosomes that could be
  charged with an amino acid and could then pass
  the amino acid to a growing polypeptide.

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tRNA structure

• Holley and et al, 1965 first determined the
  alanine tRNA structure from yeast
• The common features of tRNA (from at least 14
  tRNAs)
• Acceptor stem
• Including the two ends of the tRNA and invariant
  sequence CCA at 3 end
• D (dihydrouracil) loop
• Anticodon loop-all important
• Variable loop
• Contain 4 to 13 nt
• T loop
• T? C ? stands for pseudo-uridine

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(No Transcript)
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Recognition of tRNAs by aminoacyl-tRNA
synthetase

• The second genetic code

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AMP/amino acid coupling
AMP/tRNA displacement
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Ribosome recognize the tRNA but not the amino acid

• Lipmann, Benzer, von Ehrenstein and et al, 1962
• Cysteinyl-tRNAcys ?Alanyl-tRNAcys
• In vitro translation using poly(UGU) as
  templatethis does not contain any codon for
  alanine
• Codon for ala is GCN, cysteine is UGU
• Alanine is incorporated instead of cysteine
• Fig. 19.28

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Fig. 19.28
It is the nature of the tRNA that matters
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Given that the secondary and tertiary structures
of all tRNA are essentially the same, what base
sequences in tRNA do the synthetases recognize
when they are selecting one tRNA out of a pool of
over 20?

• The acceptor stem ?
• The anticodon ?

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Summary

• Biochemical and genetic experiments have shown
  that the anticodon, like the acceptor stem, is an
  important element in charging specificity.
• Sometimes the anticodon can be absolute
  determinant of specificity.

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Proofreading and editing by aminoacyl-tRNA
synthetases
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• 1958, Pauling used thermodynamics and found
• Ile and val only differ in CH2 group and
  one-fifth Val-tRNAile would be made
• In fact, 1/150 amino acid is activated by IleRS
  to make Val and 1/3,000 aminoacyl-tRNA is
  Val-tRNAile

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How does isoleucyl-tRNA synthetase prevent
formation of Val-tRNAIle?
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How does isoleucyl-tRNA synthetase prevent
formation of Val-tRNAIle?
Double-sieve mechanism Fersht in 1977
Fig. 19.31
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Activation site
Fig. 19.32
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Fig. 19.33
The space between Trp232 and Tyr386 is just big
enough to accommodate valine but not Ile (too
big) Abolish this region could abolish editing
but not activation activity
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Summary-1

• The amino acid selectivity of at least some
  aminoacyl-tRNA synthetases is controlled by a
  double sieve mechanism.
• The first sieve is a coarse one that excludes
  amino acids that are too big. The enzyme
  accomplishes this task with an active site for
  activation of amino acids that is just big enough
  to accommodate the cognate amino acid, but not
  larger amino acids.
• The second sieve is a fine one that degrades
  aminoacyl-AMPs that are too small.

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Summary-2

• The enzyme accomplishes this task with a second
  active site (the editing site) that admit small
  aminoacyl-AMPs and hydrolyzes them. The cognate
  aminoacyl-AMP is too big to fit into the editing
  site, so it escapes being hydrolyzed. Instead,
  the enzyme transfers the activated amino acids to
  its cognate tRNA