Karian Lee
Luther College
Decorah, IA
Faculty Mentor: Dr. James Anderson 

In all cells, translation of mRNA requires a variety of tRNAs to deliver the 20 amino acids needed to synthesize proteins. The post-transcriptional modification of nucleotides in tRNA plays an important role in their ability to function in translation. For example, modified nucleotides support the tertiary structure of tRNAs, and also play a role in the recognition of mRNA codons by tRNA anticodons. There are up to 14 post-transcriptional nucleotide modifications in every cytoplasmic tRNA. One such tRNA base modification is 1-methyladenosine (m1A), which is found predominantly at position 58 of TyC loop of tRNAinitiatorMet. We have shown that tRNAinitiatorMet is destabilized and susceptible to degradation in the absence of m1A 58. A complex composed of Gcd10p/Gcd14p was previously shown to be the enzyme responsible for the 1-methyladenosine modification at position 58 of tRNA. Based on experimental evidence, we believe the structure of this enzyme is a heterotetramer composed of two heterodimers. The goal of my project was to characterize mutations in GCD10 in order to identify amino acids that are critical for interactions between the two subunits, between the two dimers, or crucial for enzyme-substrate binding.

Random mutations of the GCD10 gene were made in an E. coli mutator strain. This library of mutants was screened in yeast for temperature sensitivity and those exhibiting a temperature-sensitive lethal phenotype at 36oC were characterized by growth phenotype, Gcd10p expression and DNA sequencing.

Growth tests showed that the mutants exhibited a temperature-sensitive lethal phenotype at 36oC but at 26oC the mutants grew similar to a strain expressing wild type Gcd10p. Gcd10p expression was tested by western blot analysis at permissive (26oC) and nonpermissive (36oC) temperatures. Several mutants were identified that expressed Gcd10p at the same level as the wild type. Genetic characterization of gcd10 mutants was carried out by DNA sequence analysis of the corresponding GCD10 gene. In one of the mutants, gcd10-5, a mutation was identified that changed the amino acid proline (422) to serine. This mutation is at a location that may be important in the quaternary structure of the enzyme. We concluded from this study that a mutation exhibiting a temperature-sensitive phenotype corresponded to an amino acid change that is predicted to disturb tetramer stability or formation.