My name is Ivailo Simoff and I am 38 years old. Currently I am at the end of my PhD period, which will end up with a thesis defence 16-06-2009. During my PhD time, my main interest was focused on ribosomal proteins (r-proteins) in S. cerevisiae. I was interested in NLS, RNA binding domains in two particular r-proteins, rpL5 and rpL15A. I would like to further broaden my knowledge and expertise in this or other field.
Abstract: In this study we provide general information on the little studied eukaryotic ribosomal protein rpL15. Saccharomyces cerevisiae has two genes, YRPL15A and YRPL15B that could potentially code for yeast rpL15 (YrpL15). YRPL15A is essential while YRPL15B is dispensable. However, a plasmid-borne copy of the YRPL15B gene, controlled by the GAL1 promoter or by the promoter controlling expression of the YRPL15A gene, can functionally complement YrpL15A in yeast cells, while the same gene controlled by the authentic promoter is inactive. Analysis of the levels of YrpL15B-mRNA in yeast cells shows that the YRPL15B gene is inactive in transcription. The function of YrpL15A is highly resilient to single and multiple amino acid substitutions. In addition, minor deletions from both the N- and C-terminal ends of YrpL15A has no effect on protein function, while addition of a C-terminal tag that could be used for detection of plasmid-encoded YrpL15A is detrimental to protein function. YrpL15A could also be replaced by the homologous protein from Arabidopsis thaliana despite almost 30% differences in the amino acid sequence, while the more closely related protein from Schizosaccharomyces pombe was inactive. The lack of function was not caused by a failure of the protein to enter the yeast nucleus.
Abstract: The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an alpha-helix with a positively charged surface that is involved in protein-5S rRNA interaction. Formation of an YrpL5.5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the alpha-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative alpha-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the alpha-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.
