2010 Rustbelt RNA Meeting
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Poster number 81 submitted by Arnab Ghosh

Truncation of Ribosomal protein S5 abrogates GCN4 expression by acting downstream of the 48S complex assembly

Arnab Ghosh (CGRHD, Dept. of BGES, Cleveland State University), Anton A. Komar (CGRHD, Dept. of BGES, Cleveland State University)

Abstract:
Ribosomal protein (rp) S5 belongs to the family of ribosomal proteins that contains rpS7 from prokaryotes and rpS5 from eukaryotes. RpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for cell viability of yeast. To investigate the function of the rpS5 and in particular the role of the N-terminal extension of the yeast protein (absent in bacteria), we obtained and characterized yeast strains in which the wt yeast rpS5 was replaced by its truncated variants, lacking 13, 24, 30 and 46 N-terminal amino acids, respectively. Biochemical analysis of the mutant yeast strains previously showed that the N-terminal part of the yeast S5 plays important roles in ensuring the efficiency and accuracy of elongation and initiation processes. In particular, we have found that cap-dependent initiation in YS5-46 strain (lacking 46 N-terminal amino acids) was reduced by about 50% as compared to the wild type strain. Here we further show that this strain fails to support re-initiation as evident by the use of GCN4-lacZ reporter constructs. Comparison of lacZ expression from various GCN4-lacZ reporter constructs (containing all four uORFs and/or two uORFs (uORFs 1&4) as well as one uORF4) allowed us to suggest that YS5-46 yeast strain possesses an initiation defect downstream of the 48S complex assembly. An increased association of initiation factors eIF2alpha, eIF1 and eIF5B with mutant 40S ribosomal subunits was also observed. We thus hypothesized, that rpS5 N-terminal truncation likely results in a slow dissociation of eIF2 from 48S complexes, thus also causing accumulation of eIF5B and in turn leading to compromised subunit joining. Therefore, truncation of rpS5 causes mutant ribosomes leaky-scan through the uORF1 AUG, making it further impossible for the scanning subunits to bypass uORFs 2, 3 or 4 and re-initiate at AUG codon 5 to translate the GCN4 ORF.

Keywords: ribosomal protein, GCN4, translation