| M W F; 10:10 - 11:00 pm |
|
Douglas W. Smith |
| York 2722 |
|
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
Readings: Brown, 10: 235-240
Outline:
A. Basic Features: nonoverlapping, commafree, degenerate / redundent , nonambiguous universal triplet Code ... [Brown, Fig 10.l6]
Examples of Codes using Nucleic Acid Sequences:
Comma code, with G as a "comma", and using several size codons (codons are in bold):
G C C G A C T G T T A C G A G A C C T A G A T G
--- ----- ------- - --------- ---
aa1 aa2 aa3 aa4 aa5 aa6
Completely overlapping comma-free triplet code (amino acids are numbered):
aa2 aa5 aa8 aa11 aa14 aa17 aa20
----- ----- ----- ----- ----- ----- -----
A G T C T T G C C A A A A G T C A G C A T T T A C C C C C G
----- ----- ----- ----- ----- ----- -----
aa1 aa4 aa7 aa10 aa13 aa16 aa19
----- ----- ----- ----- ----- ----- -----
aa3 aa6 aa9 aa12 aa15 aa18 aa21
Triplet code: a Codon is composed only of three Nucleotides ...
Degenerate or Redundant code: more than one Codon is used to encode a given Amino Acid ... [Brown, Page 235]
Note: The Genetic Code is NOT ambiguous !!
i.e., a given Codon will encode at most one amino acid.
Ambiguous code: a given Codon could encode more than one Amino Acid ...
Universal code: the same Code is used by ALL organisms
...
Generally true ... some exceptions for mitochondrial DNA, lower
eukaryotes, and few bacteria
... [Brown, Table 10.2]
B. Evidence for a commafree,
nonoverlapping Triplet code:
Crick et al rIIB proflavine genetic experiment
Used the rIIB region
of the rII gene of phage T4 ...
this is a nonessential region of the protein encoded by the rII
gene
1. Phage T4: one of the T-even bacteriophage which use
E. coli as host
Crick et al used the rIIB region
of the rII locus.
The rIIB region, at the C-terminal end of the rII locus, is nonessestial
for protein function
r+ wildtype phage: yield small plaques with fuzzy edges,
plaques that appear somewhat turbid, due to lysis inhibition
(slow lysis of infected cells)
R mutants: R = r- ... typical nomenclature used
in phage work
These mutants are rapid lysis mutants ... no lysis inhibition
in these mutants
The result is large, clear plaques with sharp edges
R mutants arise from mutations in two T4 genes, the rI and rII
genes
Growth properties on two E. coli strains, E. coli B and E. coli K12(lambda)
| E. coli B E. coli B E. coli K12(lambda)
| growth? plaques growth?
-------------|---------------------------------------------
r+ : | yes small, fuzzy yes
R = r = r- : | yes large, clear NO
Inability of T4 R mutants to grow on E. coli K12 lysogenic for Lambda but ability to grow on E. coli B is an example of phage host range mutants.
Note: Inability of T4 R mutants to grow on E. coli K12(lambda) is due to expression of the Lambda rex genes, found immediately adjacent to the CI gene and expressed from the lambda Prm promoter, in the lambda lysogen. Lambda rex mutants can be obtained that now permit growth of T4 R mutants on E. coli K12 lysogenic for the Lambda rex mutant.
2. Crick et al Experimental
Procedure and Results:
Mutagenized T4 with Proflavine: an acridine that yields
insertion-deletion mutants via intercalation between DNA base
pairs ...
Growth on E. coli B: easy to detect large clear plaques among small fuzzy ones
Obtained mutants
Obtained double mutants in rIIB, mainly via Recombination between phage DNA molecules upon "mixedly infecting" E. coli (infecting E. coli simultaneously with two different mutant T4 strains)
Two types of Double Mutants:
1) intragenic suppression ... 2nd mutation reversed effects
of 1st mutation;
2nd mutation in same gene as 1st mutation ... r+ phenotype:
small turbids
Easy to isolate, since could now form plaques on E. coli K12(lambda)
2) no suppression: still R mutant
Classified the single mutations
into two groups: called these groups + and - groups
+ mutations could suppress - mutations, vice versa
+ mutations could NOT suppress other + mutations; - mutants could
not suppress - mutations
Thus: +- doubles were often r+; ++ and -- were NEVER r+
Obtained triple mutants via
Recombination using mixed infection with a Single and a Double
mutant:
+++ and --- occasionally r+; any mixture of + and -
mutations never r+
Even went as far as: ++++++ and ------ : couple of r+ cases
Conclusion: code was a commafree, nonoverlapping, probably triplet code ...
64 codons and only 20 amino acids: code was probably degenerate ...
All of the main features of the Genetic Code ...
Main Problem: protein encoded by rIIB was unknown ! ... could not examine amino acid changes ... above expts repeated by Streisinger et al on T4 lysozyme: amino acid changes confirmed that - meant bp deletion, + meant bp insertion.
C. Codon Identifications: ... [Brown, Research Brief 10.3]
Three major in vitro protein synthesis methods used:
1. Nirenberg-Matthei and Ochoa group: use of synthetic polyribonucleotides
Used Polynucleotide phosphorylase to synthesize the synthetic mRNA species
Reaction: (a)GDP + (b)ADP + (c)UDP + (d)CDP --> [GaAbUcCd] + (a+b+c+d)Pi
Unlike DNA or RNA polymerases, no template or primer is required
Resulting synthetic RNA is nearly random in its precise sequence of the 4 bases, i.e. any NDP present in the reaction mixture is incorporated with a probability proportional to its concentration.
Now use the synthetic RNAs as mRNAs in an in vitro protein synthesis reaction.
Typical Results:
poly(U) --> poly(Phe) ... Thus: UUU -> Phe ... [Lod3, 4-28; Lod4, 4-22]
poly(UG) --> polypeptides containing Phe, Leu, Cys, Val, Trp, Gly
Problem: how to make the codon identifications for UUG, UGU, GGU, GUU, etc ???
used statistics, but polyribonucleotides synthesized were not always statistically correct ... succeeded in making about half the identifications ...
2. Khorana: synthetic RNA of KNOWN sequence
Khorana and coworkers used organic chemistry methods to synthesize RNA molecules of precisely known sequence, i.e. one now knows which base actually follows which base ... no statistics or assumptions of random incorporation are needed ...
Typical Results:
1) ACACACACACAC... --> Thr-His-Thr-His-Thr-His...
Thus: ACA & CAC -> His & Thr
BUT could not determine from this experiment which was which ...
2) AACAACAACAACAAC --> poly(Asn); poly(Thr); poly(Gln)
Thus, this synthetic mRNA yielded three different polypeptides each of which contained a single amino acid ... this was due to:
Reading Frame: in a triplet commafree nonoverlapping code, the amino acid of the resulting polypeptide depends on which nucleotide is the first nucleotide used to start the translation.
In a triplet code, there are three forward reading frames and three reverse reading frames.
Example:
Forward sequence: A G U C U U G C C A A A A G U C A G C A U U U A C C C 1st Reading Frame: ----- ----- ----- ----- ----- ----- ----- ----- ----- 2nd Reading Frame ----- ----- ----- ----- ----- ----- ----- ----- ----- 3rd Reading Frame ----- ----- ----- ----- ----- ----- ----- ----- Reverse sequence: G G G U A A A U G C U G A C U U U U G G C A A G A C U 4th Reading Frame: ----- ----- ----- ----- ----- ----- ----- ----- ----- 5th Reading Frame ----- ----- ----- ----- ----- ----- ----- ----- ----- 6th Reading Frame ----- ----- ----- ----- ----- ----- ----- -----
The codons in each of these 6 reading frames are different from each other, yielding polypeptides with different amino acid sequences.
3. Leder and Nierenberg: triplet binding assay
Charged tRNA would bind to
the small subunit Ribosome containing a given 3 base mRNA (triplet)
Such a bound charged tRNA would then stick to a filter (protein
from the small subunit ribosome present in the complex), whereas
unbound charged tRNA would pass through the filter.
Charged tRNA: tRNA with a bound amino acid
Used all 20 charged tRNA molecules in each reaction mixture, ONE of which had a radioactive amino acid ... Thus, used 20 reaction mixtures for each 3 base mRNA ... one would yield bound radioactivity to the filter ...
The 3 base mRNA triplets were synthesized using organic chemistry methods: known sequence
Thus, could identify directly which amino acid correlated with which codon or triplet
Succeeded in making all remaining codon identifications ...
D. Nature of the Genetic Code: ... [Brown, Fig 10.7]
61 Sense codons, 3 stop codons: UAG, UAA, UGA
Redundancy: 1-6 codons per amino acid
Codons for a given amino acid: variation often in 3rd position: 3rd base degeneracy
Wobble hypothesis of Crick: base pairing of the 3rd base in a codon was less stringent in its pairing with the 1st base in the tRNA anti-codon ... see tRNA discussion
AUG: both start codon and internal Methionines
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