Search

The eukaryotic Ssu72 factor is involved in several RNA biogenesis processes. It has phosphatase activity on the carboxy-terminal domain (CTD) of the major subunit of RNA polymerase II. The Kluyveromyces lactis Ssu72 (KlSsu72) shows in vitro phosphatase activity for the pNPP substrate, and this activity is inhibited by ortho-vanadate. The expression of KlSsu72 in Saccharomyces cerevisiae shows defective CTD serine5-P phosphatase activity and reveals the importance of Ssu72 for the normal CTD serine5-P levels at two growth states. The divergence is emphasised by the remarkable changes in RNA14 alternative 3'-end RNA processing, which are independent of the CTD serine5-P levels., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The Kluyveromyces lactis HIS4 gene (KlHIS4) is transcriptionally regulated by the carbon source. The promoter region encompassing positions -238 to -139 is responsible for this regulation according to lacZ reporter assays. Electrophoretic Mobility Shift Assay (EMSA) experiments on KlHIS4 promoter (positions -218 to -213, Fragment 6, F6) show a specific gel-shift band, CS1, whose intensity is carbon-source dependent in K. lactis hap2 (klhap2) knock-out strains. The klhap3 mutation is not able to cause this effect by itself, but the combination of klhap2 and klhap3 mutations has an enhanced effect on CS1 band formation. Introducing a heat shock element (HSE) at the sequence in the F6 fragment (mutated F6, F6*) increases the binding activity in the klhap2 mutant. KlHIS4 mRNA levels in the klhap2 or the double Klhap2/3p mutant do not correlate with the increase in CS1 binding activity, indicating that the factor causing CS1 is acting and only detectable in vitro. EMSA experiments with K. lactis wild-type cells under temperature stress conditions show a band enhancement (Ts1), similar in size to CS1. Cross-competition experiments between F6 and F6* show that F6* competes more efficiently than F6 for both CS1 and Ts1 formation, indicating the involvement of the HSE in the formation of the specific gel-shift bands. Moreover, the similar gel-shift patterns suggest that both bands are caused by the same heat shock-like factor under different stress conditions. Therefore, the enhancement of the CS1 band signal in the klhap2 (and klhap2/3) mutants is due to the increase in heat shock-like factors in the protein extracts from these mutant cells grown in a non-fermentable carbon source. This Klhap2-dependent stress effect was not previously described in K. lactis.

To analyze the functionality of alternative 3'-UTR processing in the yeast Kluyveromyces lactis, recombinant forms of the KlCYC1 gene containing the proximal (1-713) or the distal (699-1194) 3'-UTR region (positions related to the TAA stop codon) were obtained. The cells expressing the gene with proximal 3'-UTR showed the same growth phenotype as the wild type. When the gene expressed only the distal region, a single transcript was generated and its expression was increased in late-growth phases. Cells expressing the alternative distal 3'-UTR region showed differences in their levels of cytochrome c biomass and ethanol production with respect to the wild type. The split 3'-UTR regions were also functional as separate processing units in Saccharomyces cerevisiae. The importance of our results in recombinant gene expression applications will be discussed.

The availability of the KICYC1 sequence was used to establish homologies with other cytochrome c genes from yeasts and the fungus Neurospora crassa. In terms of nucleotide composition, the cytochrome c gene from Kluyveromyces lactis showed a higher homology with Schwanniomyces occidentalis than with Saccharomyces cerevisiae, and this point is discussed in regard to the differences found in the codon usage of these yeasts. The deduced amino acidic composition of the protein facilitated comparison of its sequence with other cytochrome c protein sequences and new assignments of phylogenetic relationships. In this context Kluyveromyces lactis was most closely related to Candida krusei.

Codon usage (CU) in Kluyveromyces lactis has been studied. Comparison of CU in highly and lowly expressed genes reveals the existence of 21 optimal codons; 18 of them are also optimal in other yeasts like Saccharomyces cerevisiae or Candida albicans. Codon bias index (CBI) values have been recalculated with reference to the assignment of optimal codons in K. lactis and compared to those previously reported in the literature taking as reference the optimal codons from S. cerevisiae. A new index, the intrinsic codon deviation index (ICDI), is proposed to estimate codon bias of genes from species in which optimal codons are not known; its correlation with other index values, like CBI or effective number of codons (Nc), is high. A comparative analysis of CU in six cytochrome-c-encoding genes (CYC) from five yeasts is also presented and the differences found in the codon bias of these genes are discussed in relation to the metabolic type to which the corresponding yeasts belong. Codon bias in the CYC from K. lactis and S. cerevisiae is correlated to mRNA levels.