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Codons with two nts specifying one aa, then 42 = 16 codons specify 20
aa
Codons with Four nts specifying one aa, then 44 = 256 codons specify
20 aa
The single and double letter codons cannot represent all of the twenty
amino acids.
2 nt deletion :
5’ ABC ABC ABC ABC ABC ABC ABC 3’
5’ ABC ABC AAB CAB CAB CAB C 3’
3 nt deletion
5’ ABC ABC ABC ABC ABC ABC ABC 3’
5’ ABC ABC ABC ABC ABC ABC 3’
The changes would be more drastic if one or two nt are deleted compared to
deletion of 3 nts. Similarly, insertions of nts. Insertion of 3 nts may change one
aa and that is perhaps more tolerable than several aa
Cell free translational systems translate given messenger RNA to the corresponding protein
in test tube reactions in the presence of a cell extract that is devoid of its DNA but contains
the necessary translational machinery like ribosomes, tRNA, amino acyl synthetases that
couple amino acids to the the respective tRNAs .
The extracts are supplemented with salts like K+ and Mg2+ , ATP, GTP etc., necessary for
protein synthesis.
To determine the amount of protein synthesis, one of the 20 amino acids will be labelled
which can be incorporated into a protein.
At the end of protein synthesis, few microliters of the reaction mixture will be spotted on a
filter paper, and is then dipped in 10% ice cold tricholoroacetic acid that precipitates protein
in the reaction mixture which is stuck to the filter paper.
Filter paper is washed with 5% hot tricholoroacetic acid (TCA) to remove any non specific
radioactivity that is bound to the filter paper. Afterwards the filters are washed with
hypochloric acid to remove any color that comes from the extract and is bound to the filter
paper (ex: in the case of red blood cell extracts)
To remove the water and dry the filters, the latter are washed with choloroform and ethanol
three-four times and are air dreied. The filters are then placed in few ml of scintilation fluid
and are read in a scintillation counter to determine the radioactivity bound to them.
Cell Free Translational Systems are used to decipher the genetic code
Cells are lysed, DNA/ nucleus is removed as it contains transcriptional
apparatus by treating the extracts with DNAses. Endogenous mRNAS can
also be removed by treating with micrococal nuclease and Ca2+ in order to
test the effect of exogenous mRNA. Prior to adding mRNA the micrococal
nuclease can be inactivated by EGTA that removes Ca2+
the cell-free protein synthesizing systems contain all the machinery viz.,
the ribosomes, transfer RNAs, aminoacyl synthetases that catalyze the
joining of amino acids to tRNAs and other soluble factors (initiation,
elongation and termination factors) required for the protein synthesis.
Additionally the extracts are supplemented with ATP, GTP, and energy
generating enzymes along with salts like K+ and Mg2+. Such cell-free
translational systems derived from bacterial extracts eventually provided a
means to determine the amino acid sequence of proteins coded by artificial
and natural templates.
The probability of any codon with two As and one C (AAC, ACA, CAA)
would be 1/5 x 1/5 x 4/5 = 4/125 = 0.032 or about 3%
Amino acid analyses of polypeptides produced by
synthetic templates
Synthetic Homopolymers and their translation yielded the
following :
AAAAAAAAAA--- Polylysine
UUUUUUUUU—polyphenylalanine
CCCCCCCCC- polyproline
GGGGGGGGG – polyglycine but synthesis is weak as
polyglycine cannot properly fold
AACAACAACAACAAC (AAC)
The common amino acid that is incorporated when the mRNAs containing
AC repeats and AAC repeats is threonine.
Triplet codon + tRNA with its amino acid + ribosomes can form a complex
and can be retained on a filter paper.
This technique is found to be relatively more informative in deciphering the
genetic code because it is easy to prepare and handle small triplet
nucleotide sequences than long RNA templates.
The synthetic triplet codons were incubated with a mixture of tRNAs each
carrying a radioactive amino acid.
Small molecules like the tRNAs carrying amino acids whose anticodons are
not complementary to the codons cannot bind to the codon in the reaction
and will pass through the filter.
Transfer RNAs with different anticodons that accept the same amino acid
are called isosccepting tRNAs. Codons specifying same aa are said to be
synonymus codons
These observations suggest that the first base in the 5’-anticodon of tRNA
does not follow strict Watson-Crick base-pair rule position while pairing with
the 3’ end of codon.
Total 64 Codons
Stop Codons Three, UAA, UAG and UGA
Sense Codons Sixty One,
Three amino acids: Leu, Ser and Arg Six Codons each, Total = 18 Codons
Five aa: Gly, Pro, Ala, Val, Thr Four codons each, Total= 20 Codons
Nine aa: Phe, Tyr, Cys, His, Glu, Gln, Asn, Two Codons each, Total = 18 Codons
Asp, Lys
Two amino acids, Met, Trp One codon each , Total = 2 Codons
Mitochondrial DNA in
Human and yeast UGA codes Trp instead as a stop codon
Human AUA codes Met instead Ile
Human AGA and AGC code Stop codon instead Arg
Nuclear DNA in
Tetrahymena UAA codes Gln instead as a stop codon
Paramecium UAG codes Gln instead as a stop codon
Mutations
Nonsense mutation
A change in the base of a codon specifying an amino acid to another base that
results in a stop codon. If it happens in the middle of a genetic message, it results
in premature termination and incomplete polypeptide synthesis.
Silent mutation
Often a change in the base that is present in the third position of a codon of an mRNA
to another base can result in a synonym that does not alter the amino acid sequence
of the encoded protein. Such changes are called silent mutations.
Neutral Mutation
It is a kind of missense mutation where the change in a base leads to a change in the
incorporation of a different amino acid that is chemically similar to the original one,
and does not influence the protein function.