1. An extensive series of mutants has been used to study the function of the U6

Question

1. An extensive series of mutants has been used to study the function of the U6 snRNA.

Each U6 mutant listed below prevents splicing. With respect to the various complexes

corresponding to each stage of splicing, determine at which point each U6 mutant will block

spliceosome assembly.

(a) U6 mutant 1: extends complementarity to U4 by 25 nucleotides

(b) U6 mutant 2: reduces complementarity to the 5’ splice site to two nucleotides

(c) U6 mutant 3: point mutation in the catalytic core

(d) U6 mutant 4: extends complementarity to U2 by 25 nucleotides

Explanation / Answer

All the U6 mutants mentioned will prevent the splicing of introns. a) U6 mutant 1: extends complementarity to U4 by 25 nucleotides. - This will effect the complex formation. b) U6 mutant 2: reduces complementarity to the 5' splice site to two nucleotides. - This affects the recognition at the 5' splice site and splicing will be inhibited. The GU sequence of the intron will not be recognized and splicing prevented. c) U6 mutant 3: point mutation in catalytic core. - Catalytic core interacts with the substrates used for two steps of self splicing reaction. If mutation takes place within it then the splicing will be affected. d) U6 mutant 4: extends complementarity to U2 by 25 nucleotides. - Binding between the U2 and branchpoint sequence of introns result in bulging out of unpaired adenosine, and initiates the nucleophilic attack on intronic 5' splice site. If, complemantarity extends then this attack will be affected and the splicing gets prevented. All the U6 mutants mentioned will prevent the splicing of introns. a) U6 mutant 1: extends complementarity to U4 by 25 nucleotides. - This will effect the complex formation. b) U6 mutant 2: reduces complementarity to the 5' splice site to two nucleotides. - This affects the recognition at the 5' splice site and splicing will be inhibited. The GU sequence of the intron will not be recognized and splicing prevented. c) U6 mutant 3: point mutation in catalytic core. - Catalytic core interacts with the substrates used for two steps of self splicing reaction. If mutation takes place within it then the splicing will be affected. d) U6 mutant 4: extends complementarity to U2 by 25 nucleotides. - Binding between the U2 and branchpoint sequence of introns result in bulging out of unpaired adenosine, and initiates the nucleophilic attack on intronic 5' splice site. If, complemantarity extends then this attack will be affected and the splicing gets prevented.

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