Poster abstracts

Poster number 39 submitted by Karthik Krishnan

Initiator tRNA-dependent and -independent interactions of Streptomyces lividans ribosomes with leaderless aph mRNA

Karthik M. Krishnan (Department of Microbiology, Miami University), Gary R. Janssen (Department of Microbiology, Miami University)

Abstract:
Streptomyces lividans ribosomes and subunits were isolated and washed with high-salt-buffer containing 1M ammonium chloride. These high-salt-washed 70S ribosomes (HS70S) and 30S subunits (HS30S) were characterized by toeprinting and filter-binding assays in the presence or absence of different combinations of purified Streptomyces initiation factors (IFs). Toeprint assays reveal that HS30S subunits exhibit tRNA-dependent binding to leaderless aph mRNA in the presence of IF2 alone or with IF1 and IF2 but not in the other possible combinations of IFs whereas HS70S ribosomes do not exhibit strong binding to leaderless aph in any possible combination of IFs. Additional analyses with filter binding assays revealed tRNA-dependent and -independent binding of HS70S ribosomes and HS30S subunits to leaderless aph mRNA with IF combinations that do not yield toeprint signals, suggesting that toeprint and filter binding assays differ with regards to the type, or strength, of complex detected. Additional characterization of the complexes is in progress, including competition assays to assess the importance of a 5’-terminal AUG in ribosome recognition of leaderless mRNA.

Keywords: Streptomyces, Translation initiation, Leaderless mRNA