PubMed · 2194105
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M Ares, A H Igel. 1990. Mutations define essential and nonessential U2 RNA structures.. https://doi.org/10.1007/bf00360444
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Despite occurring in an essential molecule, mutations in RNA polymerase readily emerge and elicit complex pleiotropic effects across different levels of biological organization, which are all modulated by environment. We investigated the impact of temperature on the effects of six mutations on sequence, structure, transcriptome, and organismal traits. We found temperature altered the transcriptomic response and key organismal traits such as growth rate and biofilm formation in a genotype-specific manner. Critically, mechanistic insights into the possible drivers of mutational effects emerged only when examining the relationships between different levels of organization: location of mutations in the tertiary structure and distance to key interacting molecules partly explained the observed transcriptomic differences, which in turn drove the impact of mutations on organismal traits. While falling short of capturing the full complexity of the system, our findings underscore the benefits of integrating insights across multiple biological levels to understand the relationship between environment and mutational effects in molecules with extensive pleiotropic effects.
The in vivo folding of subtilisin is dependent on a 77 amino acid prosequence, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme. The recent determination of the structure of a complex of the prodomain and a calcium-free subtilisin mutant has suggested how the prodomain may catalyze subtilisin folding [Bryan, P., Wang, L., Hoskins, J., Ruvinov, S., Strausberg, S., Alexander, P., Almog, O., Gilliland, G., & Gallagher, T. (1995) Biochemistry 34, 10310-10318]. In the complex, the prodomain packs against the two parallel surface helices of subtilisin (residues 104-116 and residues 133-144) and supplies caps to the N-termini of the two helices. The binding site is contained almost entirely in the linear sequence 100-144 of subtilisin. The C-terminus of the prodomain (residues 72-77) extends out from its central part to bind like a substrate in subtilisin's active site cleft. The simplest model of catalyzed folding is one in which the observed binding interaction in the complex accelerates folding by stabilizing an intermediate which includes the 45 amino acid alpha beta alpha substructure in subtilisin. According to our hypothesis, amino acids 100-144 would have a native-like fold in the intermediate which the prodomain stabilizes. Guided by the structure of the bimolecular complex of subtilisin and its prodomain, we have constructed mutations in the C-terminal region of the prodomain. Analysis of five mutants reveals a general correlation between the ability of the prodomain to bind to native subtilisin and its ability to accelerate subtilisin folding.(ABSTRACT TRUNCATED AT 250 WORDS)
A conserved helix 2 Asp is required for the proper function of many G-protein-coupled receptors. To reveal the structural basis for the role of this residue, the additive effects of mutations at this locus and at a conserved helix 7 locus were investigated in the 5-HT2A receptor. All mutant receptors studied retained high affinity agonist and antagonist binding. Whereas an Asp-->Asn mutation in helix 2 eliminated coupling, interchanging the residues at the two positions by a second mutation of Asn-->Asp in helix 7 restored receptor function. These data suggest that these residues are adjacent in space and interact. The loss of function observed with Ala at either position is consistent with each side chain forming hydrogen bonds. Molecular dynamics simulations were performed on three-dimensional computational models of agonist-receptor complexes of both the wild-type receptor and the Asp-->Asn mutant receptor. Consonant with the lack of coupling observed for the mutant construct, introducing the mutation into the computational model produced a conformational change in a direction opposite to that seen from computational simulations of activation of the wild-type receptor model. These results implicate both loci in a common hydrogen-bonding network underlying receptor activation by agonist.