María Angeles Freire-Picos

Heme-mediated transcriptional control in Kluyveromyces lactis.

Abstract Heme is of great importance in oxygen-dependent biological functions, since it serves as a prosthetic group for many proteins related to oxygen-binding, oxidative damage prevention and electron transport. It also regulates gene expression through the action of specific transcriptional regulatory factors. In this paper, we present an analysis of heme-dependent transcriptional regulation of several respiration-related genes in an aerobic respiratory yeast, Kluyveromyces lactis. We also report that the KlHEM13 gene, encoding the heme biosynthetic enzyme coproporphyrinogen oxidase, is under heme and oxygen transcriptional regulation, thereby controlling the synthesis of the effector, heme. KlHEM13 is induced during hypoxia, which represents the first report of a transcriptionally regulated gene with this behaviour in K. lactis.

Haem regulation of the mitochondrial import of the Kluyveromyces lactis 5-aminolaevulinate synthase: an organelle approach

The enzyme 5‐aminolaevulinate acid synthase (ALAS) catalyses the first reaction in the haem biosynthetic pathway. In eukaryotes this protein is translated by cytosolic ribosomes and then targeted to the mitochondria. We present evidence that in the yeast Kluyveromyces lactis haem exerts a feedback control upon the import of the ALAS into mitochondria. The ALAS from K. lactis (KlALAS) contains two haem regulatory motifs (HRM) in the mitochondrial targeting signal. Mutagenesis experiments reveal the involvement of these HRM in the response of the KlALAS to haem. Copyright

Involvement of Pta1, Pcf11 and a KlCYC1 AU-rich element in alternative RNA 3'-end processing selection in yeast.

This work reports the involvement of yeast RNA processing factors Pta1 and Pcf11 in alternative 3′‐end RNA processing. The pta1‐1 and pcf11‐2 mutations changed the predominance of KlCYC1 1.14 and 1.5 kb transcript isoforms. Mutation of the KlCYC1 3′‐UTR AU‐rich sequence at positions 679–690 (mutant M1) altered transcript predominance. Moreover, expression of M1 in the yeast mutants partially suppressed their effects in the predominance pattern. The combination of the M1 and M2 (694–698 deletion) mutations abolished the alternative processing. Pta1 involvement in this selection was confirmed using the Pta1‐td degron strain.

The HIS4 gene from the yeast Kluyveromyces lactis

The Kluyveromyces lactis HIS4 gene was cloned by complementation of a Saccharomyces cerevisiae his4 mutant. Sequence analysis revealed a 2388 bp open reading frame encoding a single polypeptide predicted to encompass three distinct enzymatic activities (phosphoribosyl‐AMP cyclohydrolase, phosphoribosyl‐ATP pyrophosphohydrolase and histidinol dehydrogenase). This structural organization is strikingly similar to that of the His4 proteins from S. cerevisiae and Pichia pastoris. Transcript analysis detected a single mRNA species of 2.5 kb. The EMBL accession number of this gene is Y09503.

PICDI, a simple program for codon bias calculation

PICDI is a very simple program designed to calculate the Intrinsic Codon Deviation Index (ICDI). The program is available in Macintosh as well a PC format. Requirements for correct input of the sequences have been kept to a minimum and the analysis of sequences up to 2000 codons is very quick. The ICDI is very useful for estimation of codon bias of genes from species in which optimal codons are not known. The availability of a computer program for its calculation will increase its usefulness in the fields of Molecular Biology and Biotechnology.

Transcriptional repression by Kluyveromyces lactis Tup1 in Saccharomyces cerevisiae

The general repression complex, constituted by the yeast Tup1 and Ssn6 factors, is a conserved global regulator of transcription in eukaryotes. In the yeast Saccharomyces cerevisiae, it is an important repressor of hypoxic genes, such as ANB1, under aerobic conditions and deletion of the TUP1 gene causes a flocculation phenotype. The KlTUP1 gene from the yeast Kluyveromyces lactis encodes for a protein with 83% similarity to Tup1 in S. cerevisiae. Despite the general domain conservation, the database searches showed the absence of a characteristic Tup1 glutamine-rich domain (Q1 at positions 96–116). Instead, there was a non-conserved sequence lacking the α-helix structure in this region. The ability to act as a transcriptional repressor was tested by expressing the KlTUP1 gene, in both high- and low-copy vectors, in an S. cerevisiae tup1 mutant strain. Repression effects were studied using the aerobic repressible reporter ANB1–lacZ and the effect on flocculation. In both regulatory systems, low levels of KlTup1 caused moderate (~30%) repression, but when the number of KlTup1 copies was increased, only the ANB1 reporter raised the repression levels of S. cerevisiae Tup1. These results show the capability of KlTup1 to act as a repressor in S. cerevisiae. The lower repression reached in S. cerevisiae is discussed in terms of structural differences.

Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast.

Many eukaryotic genes undergo alternative 3′-end poly(A)-site selection producing transcript isoforms with 3′-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3′-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3′-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

A stress response related to the carbon source and the absence of KlHAP2 in Kluyveromyces lactis

The Kluyveromyces lactisHIS4 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. lactishap2 (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.

Structurally conserved and functionally divergent yeast Ssu72 phosphatases.

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.

Alternative Polyadenylation in Yeast: 3 ́-UTR Elements and Processing Factors Acting at a Distance

One of the key steps necessary to obtain a messenger RNA (mRNA) is 3 -end RNA processing. The two specific 3 -end processing reactions for genes transcribed by RNAP-II (RNA polymerase II) are pre-mRNA cleavage followed by the addition to the cleaved 3 -end of a polyadenine “tail” (polyadenylation) catalyzed by the poly-A polymerase. For some genes this 3 -end processing gets more complex due to the fact that a single gene can be transcribed in two or more mRNAs differing in their 3 -UTR length due to the presence of two or more cleavage and polyadenylation points. This multiple (or alternative) 3 -end processing is also referred to as alternative polyadenylation (APA). In this chapter we will analyze some aspects of alternative RNA processing in eukaryotes using yeast as model. Hence, we will focus on the involvement of RNA processing machinery factors and elements in the UTRs (3 -UnTranslated Regions) necessary for this processing.