Yeast strains with N-terminally truncated ribosomal protein S5: implications for the evolution, structure and function of the Rps5/Rps7 proteins

Abstract

Ribosomal protein (rp)S5 belongs to the family of the highly conserved rp’s that contains rpS7 from prokaryotes and rpS5 from eukaryotes. Alignment of rpS5/rpS7 from metazoans (Homo sapiens), fungi (Saccharomyces cerevisiae) and bacteria (Escherichia coli) shows that the proteins contain a conserved central/C-terminal core region and possess variable N-terminal regions. Yeast rpS5 is 69 amino acids (aa) longer than the E. coli rpS7 protein; and human rpS5 is 48 aa longer than the rpS7, respectively. To investigate the function of the yeast rpS5 and in particular the role of its N-terminal region, we obtained and characterized yeast strains in which the wild-type yeast rpS5 was replaced by its truncated variants, lacking 13, 24, 30 and 46 N-terminal amino acids, respectively. All mutant yeast strains were viable and displayed only moderately reduced growth rates, with the exception of the strain lacking 46 N-terminal amino acids, which had a doubling time of about 3 h. Biochemical analysis of the mutant yeast strains suggests that the N-terminal part of the eukaryotic and, in particular, yeast rpS5 may impact the ability of 40S subunits to function properly in translation and affect the efficiency of initiation, specifically the recruitment of initiation factors eIF3 and eIF2.

Rps5-Rps16 communication is essential for efficient translation initiation in yeastS. cerevisiae

“…Our biochemical analysis suggests that truncation of the Rps5 N-terminal region, or mutation of an NTD residue (K45) that contacts Rps16, compromises hydrolysis of eIF2-bound GTP (in the 48S PIC), increasing accumulation of eIF1 and eIF5B (in addition to the eIF2 accumulation noted before (10)) while reducing association of eIF5, thereby delaying subunit joining and progression of the 80S ribosome to the elongation-competent state. Remarkably, similar effects were observed on eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA when base-paired to AUG in the P site (2,3,12).…”

“…rps5-Δ0 , rps5-Δ1-13 , rps5-Δ1-24 , rps5-Δ1-30 and rps5-Δ1-46 strains (Table 1) have been previously described (10), in which the chromosomal RPS5 gene is deleted and replaced with a kanMX cassette and mutant or wild type (WT) RPS5 alleles are present on high-copy plasmids and expressed from the strong S. cerevisiae TEF1 promoter. The following strains of a similar design, rps5-K41A Mat a his3-1, leu2-0, ura3-0 , rps5::kanMX , < yrps5-K41A ; LEU2, 2μ >, rps5-F43G Mat a his3-1, leu2-0, ura3-0 , rps5::kanMX , < yrps5-F43G ; LEU2, 2μ >, rps5-K45A Mat a his3-1, leu2-0, ura3-0 , rps5::kanMX , < yrps5-K45A ; LEU2, 2μ >, expressing yeast rps5 variants with the indicated amino acid substitutions, were obtained as follows: point mutations were first introduced into the RPS5 gene in pTEF_yS5 plasmid (10) using side-directed mutagenesis and the following primers 5′-CAAACCGAGATTGCGTTGTTCAAC-3′ forward and 5′-GTTGAACAACGCAATCTCGGTTTG-3′ reverse (pTEF_yS5-K41A); 5′-GAGATTAAGTTGGGCAACAAATGGTC-3′ forward and 5′-GACCATTTGT TGCCCAACTTAATCTC-3′ reverse (pTEF_yS5-F43G); and 5′-AGTTGTTCAACGCATGGTCTTTTG-3′ forward and 5′-CAAAAGACCATGCGTTGAACAACT-3′ reverse (pTEF_yS5-K45A).…”

“…The following strains of a similar design, rps5-K41A Mat a his3-1, leu2-0, ura3-0 , rps5::kanMX , < yrps5-K41A ; LEU2, 2μ >, rps5-F43G Mat a his3-1, leu2-0, ura3-0 , rps5::kanMX , < yrps5-F43G ; LEU2, 2μ >, rps5-K45A Mat a his3-1, leu2-0, ura3-0 , rps5::kanMX , < yrps5-K45A ; LEU2, 2μ >, expressing yeast rps5 variants with the indicated amino acid substitutions, were obtained as follows: point mutations were first introduced into the RPS5 gene in pTEF_yS5 plasmid (10) using side-directed mutagenesis and the following primers 5′-CAAACCGAGATTGCGTTGTTCAAC-3′ forward and 5′-GTTGAACAACGCAATCTCGGTTTG-3′ reverse (pTEF_yS5-K41A); 5′-GAGATTAAGTTGGGCAACAAATGGTC-3′ forward and 5′-GACCATTTGT TGCCCAACTTAATCTC-3′ reverse (pTEF_yS5-F43G); and 5′-AGTTGTTCAACGCATGGTCTTTTG-3′ forward and 5′-CAAAAGACCATGCGTTGAACAACT-3′ reverse (pTEF_yS5-K45A). The resultant pTEF_yS5 plasmids carrying mutant RPS5 were transformed into the heterozygous diploid BY4743 [4741/4742] MAT a /MATα, his3-1/his3-1, leu2-0/leu2-0, lys2-0/+, met15-0/+, ura3-0/ura3-0, RPS5/rps5::kanMX .…”

“…The protein forms part of the exit (E) site, is essential for cellular viability and recent data suggest that yeast Rps5 functions in translation initiation (9,10) as well as 40S head formation (11). Rps5/S7 proteins possess conserved central and C-terminal regions and exhibit variability at the N-terminus, with fungi and fruit flies exhibiting the longest N-terminal tail regions as compared to bacteria and metazoans.…”

Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

“…30 This observation is supported by genetic analyses in yeast showing that truncation of the uS7 N-terminus results in depletion of eIF3 from the initiation complex. 57 Very recently, Erzberger and coworkers presented an X-ray structure of the universally conserved core subunits of eIF3 (at »3.5 A ) and modeled the eIF3 complex position on the yeast 40S ribosomal subunit by employing cryo-EM and cross-linking of the factor (s) followed by mass spectrometry (MS). 58 This reconstruction provided evidence supporting some of the previously reported interactions (between eIF3 and eukaryote-specific 40S regions) observed by biochemical, genetic and structural analyses, but also revealed a number of new/ alternative interactions.…”