Mutational Event, Wild type enzyme Sequence - (Feb/05/2002 )

Any body answer has for this? Me struggling for this. Let me know please.

A few amino acids are removed from the C-terminal end of the beta-lactamase enzyme from B. lichenformis after it is synthesized. The sequence of the C-terminus of the enzyme can be deduced by comparing it to a mutant in which the reading frame is shifted by insertion or deletion of a nucleotide. The amino acid sequence of the purified wild-type enzyme and that from the frameshift mutant are given below, from amino acid residue 263 to the C-terminal end.

wild type:  NMNGK
mutant:  NMIWQICVMKD

a.) What was the mutational event that gave rise to the frameshift mutant?

b.) Deduce the number of amino acids in the synthesized form of the wild -type enzyme and, as far as possible, the actual sequence of the wild-type enzyme.

buh

Ok,

you first have to deduce the nucleic-acid sequence from the peptides that can rise both of them when a frameshift occures. I.e. you are searching for a sequence that can be NGK or shifted IWQ. So, how do we get from N to I?

N is either AAC or AAT while

I is ATA, ATC or ATT.

So we could go from N to I with an deletion of the second A of AAT, if the first base of the following triplet would be a A,C or T. Since it's a G(glycine) and Gs triplett are GGN, a deletion is not possible or the sequence would be ATG (=M) and not I. So a Insertion has to have occured.

AATGGN -> ATATGGN

the N here has to be a C, BTW, since only then the sequence can go on in the way you mentioned.

From here you can go on an reverse translate the aminoacids from the mutant to DNA, but beware of the ambiguity!

Because of the abmiguity, once can only tell that it have been 3 more aminoacids in the wt since then a stop codon (TGA) happens to lay on the way. And even so, there's no possibility to say something about the original sequence further than you have given, since when you back-calulate the original sequence from the frameshift, the wobble (ambiguity) in the third base moves into second place in each triplett. So you just can tell anymore.

The sequences as deducible:


wildtype:
AAT ATG AAT GGC AAA THT GYG TNA TGA ARG AY
N...M...N...G...K...?...?...?...Stop
mutant:
AAT ATG ATA TGG CAA ATH TGY GTN ATG AAR GAY
N...M...I...W...Q...I...C...V...M...K...D

mike