Prokaryotic Translation Initiation Factor-2 Domains Responsible for 30 S Ribosomal Subunit Recognition (2004)

Undergraduate: Pamela Scott

Faculty Advisor: Linda Spremulli
Department: Chemistry

---

The goal of this project is to investigate the mechanism of the initiation of protein synthesis in prokaryotes and to examine the differences between this mechanism and that of eukaryotic organellar translation. Initiation factor-2 (IF-2) plays a major role in the initiation phase of protein synthesis by promoting the binding of the initiator tRNA, fMet-tRNAfmet, to the 30 S ribosomal subunit. Domains I and II of E. coli IF-2 have been shown to specifically interact with the 30 S ribosomal subunit in the absence of additional factors. In order to verify this observation, the genetic information for domains I and II of E. coli IF-2 was amplified using the polymerase chain reaction (PCR) and cloned into the vector pET21. This DNA sequence was engineered to include a 10 amino acid c-myc tag located at the C-terminus for detection through immunological analysis. E. coli IF-2 domains I and II was over-expressed and purified to near homogeneity using Ni-NTA affinity chromatography to verify that these domains specifically interact with the 30 S ribosomal subunit. This project also includes the investigation of why mitochondrial IF-2 (IF-2mt) is active on mitochondrial 55 S ribosomes as well as E. coli 70 S ribosomes, but E. coli IF-2 is not active on mitochondrial 55 S ribosomes and fails to stimulate fmet-tRNAfMet binding to the 55 S ribosomes. E. coli IF-2 consists of domains I through VI while IF-2mt consists only of the corresponding domains III through VI. It is hypothesized that domains IV through VI of E. coli IF-2 will be active on 55 S mitochondrial ribosomes since domains IV and VI are highly conserved in E. coli and mitochondrial IF-2. An expression construct of domains IV through VI of E. coli IF-2 will be prepared to see if these domains are active on 55 S mitochondrial ribosomes.