TY - JOUR
T1 - Sequence specificity of the P6 pairing for splicing of the group I td intron of phage T4
AU - Ehrenman, Karen
AU - Schroeder, Renee
AU - Chandry, P. Scott
AU - Hall, Dwight H.
AU - Belfort, Marlene
N1 - Funding Information:
We wish to thank our colleagues in the laboratory for their helpful input into this work, particularly Jill Galloway Salvo for useful discussions and William Chan and Debbie Bell Pedersen for moral support to K.E. in the final stages of her Ph.D. research. We are thankful to Jonathan Clyman, Susan Quirk and Jill Galloway Salvo for critical reading of the MS and Carolyn Wieland for expert preparation of the MS. We thank Francois Michel and colleagues for permission to cite their unpublished results. This work was supported by NSF grant DMB8502961 and NH grant GM39422 to M.B., NIH grant GM36714 to D.H.H., and Fonds zur Forderung der wissenschaftlichen Forschung, Vienna, Austria to R.S.
PY - 1989/11/25
Y1 - 1989/11/25
N2 - Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme. All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure. Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5′ element (P6[5′] of the putative two-base-pair P6 stem. An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6[5′] and P6[3′] compensatory mutations. Second, all P6[5′] and P6[3′] mutants are defective in the first step of splicing, guanosine dependent 5′ splice site cleavage, whereas their activity at the 3′ splice site is variable. Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem. Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent. Restoration of phenotype of the compensatory P6[5′]:[3′] constructs is weak under stringent in vitro conditions as well as in vivo. This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements.
AB - Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme. All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure. Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5′ element (P6[5′] of the putative two-base-pair P6 stem. An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6[5′] and P6[3′] compensatory mutations. Second, all P6[5′] and P6[3′] mutants are defective in the first step of splicing, guanosine dependent 5′ splice site cleavage, whereas their activity at the 3′ splice site is variable. Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem. Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent. Restoration of phenotype of the compensatory P6[5′]:[3′] constructs is weak under stringent in vitro conditions as well as in vivo. This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements.
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U2 - 10.1093/nar/17.22.9147
DO - 10.1093/nar/17.22.9147
M3 - Article
C2 - 2685756
AN - SCOPUS:0024345294
SN - 0305-1048
VL - 17
SP - 9147
EP - 9163
JO - Nucleic acids research
JF - Nucleic acids research
IS - 22
ER -