Section 3. Transcription, RNA modification and translation 3.7: Genetic Code

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Section 3. Transcription, RNA modification and
translation3.7 Genetic Code
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3 nucleotides encode a single amino acid

• For 4 nucleotides (ATGC) to encode 20 amino
  acids, you need a coding unit of at least 3
• A coding unit of 2 nucleotides can only encode 16
  amino acids (4x4)
• A coding unit of 3 nucleotides can only encode 64
  amino acids (4x4x4)
• Insertions or deletions of 1, 2, 4, 5, etc
  nucleotides cause a severe loss of function
  resulting from a change in the open reading
  frame, but insertions or deletions of 3, 6, 9,
  (3n) have little effect on the phenotype, because
  the open reading frame is not affected for most
  of the mRNA
• History of how codons are decoded p. 1287-1289

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Table 32-2 The Standard Genetic Codea.
Of the 64 codons, 61 specify amino acids and the
other 3 are signals to terminate translation
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Degeneracy of the code

• Degeneracy refers to the fact that almost all
  amino acids are encoded by multiple codons.
• Degeneracy is found primarily in the 3rd position
  of the codon, i.e. the nucleotide in the 3rd
  position can change without changing the amino
  acid specified.
• In some cases, the 1st position is also
  degenerate.

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Codons for initiation of translation

• Major codon for initiation is AUG
• Regardless of codon used, the first amino acid
  incorporated in E. coli is formyl-Met.
• For the 4288 genes identified in E. coli

AUG is used for 3542 genes. GUG is used for 612
genes. UUG is used for 130 genes. AUU is used
for 1 gene. CUG may be used for 1 gene.
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Codons for termination of translation

• UAA (ochre), UAG (amber), UGA (opal)
• For the genes identified in E. coli

UAA is used for 2705 genes. UGA is used for
1257 genes. UAG is used for 326 genes.
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Genetic code is univeral (almost)

• All organisms so far examined use the code as
  originally deduced
• We can produce a human protein by using E. coli
  over-expression system!
• The standard genetic code is widespread but not
  universal, e.g. in RNA derived from mitochondrial
  DNA (Table 32-3)

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Differential codon usage

• Some codons are used much more frequently than
  others to encode a particular amino acid.
• The pattern of codon usage varies between species
  and even among tissues within a species.
• Correlates with tRNA abundance.
• Pattern of codon usage can be a predictor of
  level of expression of a gene.
• Preferred codon usage is a help in reverse
  genetics.

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Differential codon usage 2

• Codon usage database http//www.kazusa.or.jp/codo
  n/

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Three possible reading frames
Mol. Biol. Gene, Fig. 14-1
Some phage DNA segments contain overlapping genes
in different reading frames (Fig. 32-6) Maximal
use of the little DNA that they can pack inside
their capsids
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Wobble in anticodon-codon pairing

• Some nucleotides in the 1st position of the
  anticodon (in tRNA) can pair with gt1 nucleotide
  in the 3rd position of the codon (in mRNA)
• G can pair with U and I can pair with U, C or A.
• Result 61 codons can read by as few as 31 tRNAs

Table 32-5 Allowed Wobble Pairing Combinations
in the Third CodonAnticodon Position.
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Types of mutations in coding regions

• Silent (synonymous)
• Do not change the encoded amino acid
• Occur in degenerate positions in the codon
• Are often not subject to purifying selection and
  thus occur more frequently in evolution
• Nonsilent (nonsynonymous)
• Do change the encoded amino acid
• Occur in non-degenerate positions in the codon
• Are more likely to be subject to purifying
  selection and thus occur less frequently in
  evolution

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Changes that alter the encoded product

• Missense cause a replacement of an amino acid
• e.g. CAA (Gln) -----gt CGA (Arg)
• Nonsense cause a termination of translation
• e.g. CAA (Gln) -----gt UAA (term)
• Frameshift insertion or deletion that changes
  the reading frame
• e.g. CAA (Gln) -----gt C-A (frameshift)

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Suppressor mutations

• Mutations at a second site that can overcome the
  effects of a missense or nonsense mutation are
  suppressors.
• Can be in the same gene (but affecting a
  different codon) OR can be in a different gene.
• Isolation of suppressor mutations in other genes
  indicates that the product of the other gene
  interacts with the product encoded by the gene
  with the original mutation.

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Nonsense Suppression

• Amber (UAG), ochre (UAA), opal (UGA)
• tRNA mutation recognizes a nonsense (stop) codon
  (Table 32-6)
• How do cells tolerates a mutation that both
  elimination of normal tRNA and prevents the
  termination of protein synthesis?
• Mutated tRNA is usually minor member of a set of
  tRNAs
• Many mRNA have two tandem stop codons

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Quiz

• 1. Which of the following mutations could occur
  by a single nucleotide substitution?
• 1.1 Phe to Leu
• 1.2 Lys to Ala
• 1.3 Ala to Thr
• 2. A codon for Lys can be converted by a single
  nucleotide substitution to a codon for Ile. What
  is the sequence of the original codon for Lys?

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Components needed for Translation

• tRNAs
• Aminoacyl-tRNA synthetases
• Ribosomes

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Figure 32-7 The adaptor hypothesis. It
postulates that the genetic code is read by
molecules that recognize a particular codon and
carry the corresponding amino acid.
Transfer RNAs tRNAs serve as adapters
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Primary structure of tRNAs

• Short 54 to 100 nucleotides long (most have
  76)
• Have a CCA at their 3 end
• - bacteria CCA is coded on DNA
• - eukaryote CCA is added by enzyme
• A charged tRNA has an amino acid attached to its
  3 end (2-OH or 3-OH group).
• Have a large number of modified bases
• Reduction of a double bond in uridine gives
  dihydrouridine (D)
• leads to the name D-loop in tRNA
• In pseudouridine, carbon at position 5 is
  replaced by a nitrogen, abbreviated y .
• The nucleotide triplet TyC is characteristic of
  another loop in tRNA.
• All 4 bases can be methylated

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Secondary structure of tRNA

• Cloverleaf 4 arms (duplex RNA) and 3 loops
• Amino acid acceptor arm duplex between the 5
  segment and 3 segment, but terminal CCA is not
  base paired
• D arm ends in D loop
• Anticodon arm ends in anticodon loop anticodon
  is in the center of the loop
• TyC arm ends in TyC loop.
• Variable loop just before TyC arm

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Tertiary structure of tRNA

• Fat L
• Base pairing between nucleotides in the D loop
  and the TyC loop, plus other interactions pull
  the tRNA cloverleaf into a pseudoknot.
• Two RNA duplexes predominate in the fat L
  structure
• TyC stem is continuous with the amino acid
  acceptor stem one arm of the L
• D stem is continuous with the anticodon arm
  other arm of the L
• Amino acid acceptor site is maximally separated
  from the anticodon.

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Figure 32-11b Structure of yeast tRNAPhe. (b)
The X-ray structure drawn to show how its base
paired stems are arranged form the L-shaped
molecule.
Figure 32-9 Cloverleaf secondary structure of
tRNA.
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Attachment of amino acids to tRNA Aminoacyl-tRNA
synthetases

• 20 enzymes, 1 per amino acid
• Highly specific on BOTH business ends of the
  tRNA
• Each must recognize several cognate tRNAs
• Recognize several or all the tRNAs whose
  anticodons complement the codons specifying a
  particular amino acid
• Must recognize the correct amino acid
• Two different classes of aminoacyl-tRNA
  synthetases, based on 3D structure

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Mechanism of aminoacyl-tRNA synthetases

• 2-step reaction
• 1st Amino acid is activated by adenylylation
• 2nd Amino acid is transferred to to the 3 or 2
  OH of the ribose of the terminal A on tRNA
• Product retains a high-energy bond joining the
  amino acid to the tRNA
• Unusual in that this is an ester linkage
• Provides the thermodynamic energy to drive
  protein synthesis
• Hydrolysis of PPi to 2 Pi can drive the synthesis
  of aminoacyl-tRNA

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Addition of amino acids to tRNAs Step 1
Step 1
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Addition of amino acids to tRNAs Step 2
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Figure 32-21 X-Ray structure of T. thermophilus
isoleucyltRNA synthetase in complex with tRNAIle
and mupirocin.
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Proofreading by aminoacyl-tRNA synthetases

• In addition to precision in the initial
  recognition of substrate amino acids, the aa-tRNA
  synthetases catalyze proofreading reactions.
• If an incorrect amino acid is used in the
  synthetase reaction, it can be removed.
• Some enzymes check the amino acid at the
  aminoacyl-adenylate intermediate. If incorrect,
  this intermediate is hydrolyzed.
• Other enzymes check the aminoacyl-tRNA product,
  and cleave off incorrect amino acids.

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Figure 32-22b Cartoon comparison of the
putative aminoacylation and editing modes of
IleRS tRNAIle.
Figure 32-23 Schematic diagram of the
aminoacylation and editing mechanisms of Class I
and Class II aaRSs emphasizing the mirror
symmetry of their overall mechanisms.
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Anticodon determines specificity

• Does a ribosome recognize the anticodon on the
  tRNA or the amino acid?

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Special tRNA for initiation of translation

• fmet-tRNAf met is used at initiation codons (AUG,
  GUG, UUG )
• Carries formylmethionine, or fmet (blocks the
  amino terminus)
• fmet is the initiating amino acids in bacteria,
  but methionine is used in eukaryotes
• In both cases, a special initiating tRNA is used.
• met-tRNAm met is used at internal codons.
• Different amino acids are used, depending on the
  context.