The amino acid sequence of polypeptides is coded for by genes. concept of a colinear relationship between gene structure and protein clusive evidence was presented for the colinearity of a segment of the A gene and. Presentation on theme: "Colinearity of Gene and Protein"— Presentation transcript: Molecular Basis for Relationship between Genotype and Phenotype .
In the early s four groups were working on the problem. The key to winning the race to prove colinearity was to use a system where we could quickly and efficiently generate a map of genetic lesions as well as a map of amino acid changes in the corresponding proteins. We realized that we could accomplish this using the trpA gene and TrpA protein of E.
The unique feature of the TrpA protein is that it is a component of an enzyme complex. TrpA and TrpB subunits together form the enzyme tryptophan synthase. Figure 3 shows that each subunit has a distinct enzymatic activity but the physical presence of each subunit is required for the other to function.
Colinearity of Gene and Protein - ppt video online download
Thus TrpA proteins with missense mutations amino acid substitutions are enzymatically inactive in the TrpA reaction but they activate the TrpB subunit of the enzyme complex. This provided us with a simple enzymological assay for missense TrpA proteins. We determined the positions of the amino acid changes in different purified missense TrpA proteins using the "peptide fingerprinting" approach introduced by Ingram Ingram, Separately a fine structure genetic map of trpA was prepared, using phage mediated transduction.
This map related the sites mutated in our set of trpA missense mutants. The map was based on the recombination frequencies detected in pairwise transduction crosses, and by the use of an outside genetic marker to establish the relative order of any two mutationally altered sites, a process known as three-factor mapping.
Three-factor mapping allows detection of rare double recombination events. Figure 4 shows the principle of using an outside heterozygous marker. Which outside allele is associated with wild-type recombinants will depend on the order of the mutations in the gene. In bacterial recombination, because one of the DNAs is provided as a fragment, one recombination event occurs between the two mutations of interest and the second occurs to one side of them.
When we compared the positions of mutations on the genetic map of the trpA gene and the positions of corresponding amino acid changes in the TrpA protein we found that the farther apart two mutations were in map units, the more amino acids there were between the substitutions in the polypeptide.
Figure 5 shows how the co-linearity of gene and protein was established. Two years later the complete sequence of the TrpA protein was determined and colinearity was demonstrated for the entire gene and protein Yanofsky et al.
Frederick Sanger worked out a brilliant method for deducing the sequence of large polypeptides. There are several different proteolytic enzymes—enzymes that can break peptide bonds only between specific amino acids in proteins.
Proteolytic enzymes can break a large protein into a number of smaller fragments, which can then be separated according to their migration speeds in a solvent on chromatographic paper. Because different fragments will move at different speeds in various solvents, two-dimensional chromatography can be used to enhance the separation of the fragments Figure When the paper is stained, the polypeptides appear as spots in a characteristic chromatographic pattern called the fingerprint of the protein.
Each of the spots can be cut out, and the polypeptide fragments can be washed from the paper. Because each spot contains only small polypeptides, their amino acid sequences can be easily determined. Figure Two-dimensional chromatographic fingerprinting of a polypeptide fragment mixture. A protein is digested by a proteolytic enzyme into fragments that are only a few amino acids long. A piece of chromatographic filter paper is then spotted with this mixture more Using different proteolytic enzymes to cleave the protein at different points, we can repeat the experiment to obtain other sets of fragments.
The fragments from the different treatments overlap, because the breaks are made in different places with each treatment. The problem of solving the overall sequence then becomes one of fitting together the small-fragment sequences—almost like solving a tricky jigsaw or crossword puzzle Figure Figure Alignment of polypeptide fragments to reconstruct an entire amino acid sequence.
This condition known as alkaptonuria happens when there is a buildup of the chemical homogentisate, which causes the darkening of urine. In most situations, excess amounts of amino acid phenylalanine are metabolised by the body. This led Garrod to surmise that the enzyme responsible for its breakdown must be defective in these patients. In addition, since the black urine phenotype was passed from generation to generation in a regular pattern, Garrod reasoned that a gene had to be responsible for the production of the defective enzyme.
He attributed a defective enzyme to a defective gene, suggesting a direct link between genes and proteins. The Relationship Between Genes and Proteins Most genes contain the information require to make proteins. The journey from gene to protein is one that is complex and controlled within each cell and it consists of two major steps — transcription and translation. Together, these two steps are known as gene expression.