François Lacroute

Genetical aspects of [URE3], a non-mitochondrial, cytoplasmically inherited mutation in yeast

Summary[URE3], a non-mitochondrial non-mendelian mutation which modifies drastically yeast nitrogen metabolism has been genetically studied. Cytoduction experiments show definitely that the inheritance of the determinant is not linked to the nucleus. The maintenance of the [URE3] determinant seems controlled by the product of a conventional nuclear gene (ure2) which is itself involved in nitrogen metabolism. The (ure2) mutation alone gives the same phenotype as [URE3] but it is impossible to obtain a stable recombinant containing simultaneously the (ure2) mutation and the [URE3] determinant. Application of the Newcombe respreading experiment demonstrates that the [URE3] mutational event occurs before the selection procedure and is therefore not strictly adaptative. Nevertheless, the nature of the selection medium changes considerably the frequency of the [URE3] mutants recovered.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

Futile cycle of transcription initiation and termination modulates the response to nucleotide shortage in S. cerevisiae.

Hidden transcription in eukaryotes carries a large potential of regulatory functions that are only recently beginning to emerge. Cryptic unstable transcripts (CUTs) are generated by RNA polymerase II (Pol II) and rapidly degraded after transcription in wild-type yeast cells. Whether CUTs or the act of transcription without RNA production have a function is presently unclear. We describe here a nonconventional mechanism of transcriptional regulation that relies on the selection of alternative transcription start sites to generate CUTs or mRNAs. Transcription from TATA box proximal start sites generates unstable transcripts and downregulates expression of the URA2 gene under repressing conditions. Uracil deprivation activates selection of distal start sites, leading to the production of stable mRNAs. We describe the elements that govern degradation of the CUT and activation of mRNA production by downstream transcription initiation. Importantly, we show that a similar mechanism applies to other genes in the nucleotides biogenesis pathway.

Organization of the yeast URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetase-aspartate transcarbamylase complex.

The 6636 bp of the yeast URA2 gene encoding the carbamoylphosphate synthetase-aspartate transcarbamylase complex have been sequenced. The protein is organized into four regions, three of which are functional domains as indicated previously by genetic analysis. The fourth domain corresponds to a defective dihydroorotase called DHOase-like. The URA2 gene complex with the same organization as the equivalent genes in higher eukaryotes suggests an evolution from a common ancestral gene.

A general suppressor of RNA polymerase I, II and III mutations in Saccharomyces cerevisiae

A multicopy genomic library of Saccharomyces cerevisiae (strain FL100) was screened for its ability to suppress conditionally defective mutations altering the 31 kDa subunit (rpc31–236) or the 53 kDa subunit (rpc53-254/424) of RNA polymerase III. In addition to allele-specific suppressors, we identified seven suppressor clones that acted on both mutations and also suppressed several other conditional mutations defective in RNA polymerases I or II. All these clones harbored a complete copy of the SSD1 gene. The same pleiotropic suppression pattern was found with the dominant SSD1-v allele present in some laboratory strains of S. cerevisiae. SSD1-v was previously shown to suppress mutations defective in the SIT4 gene product (a predicted protein phosphatase subunit) or in the regulatory subunit of the cyclic AMP-dependent protein kinase. We propose that the SSD1 gene product modulates the activity (or the level) of the three nuclear RNA polymerases, possibly by altering their degree of phosphorylation.

Construction of a Yeast Strain Deleted for the TRP1 Promoter and Coding Region that Enhances the Efficiency of the Polymerase Chain Reaction‐Disruption Method

The sequence of the genome of Saccharomyces cerevisiae was recently determined. As well as all the information concerning the structure of the chromosomes the scientific community had to deal with the discovery of dozens of new open reading frames (ORFs) of unknown function. The study of these ORFs requires the development of simple procedures that can be used on a large scale. In the framework of a European Pilot Project we have described a new approach for deleting ORFs. This method is based on transformation with a polymerase chain reaction product but is limited by the use of a strain deleted for the auxotropic marker. We present here the construction of a new recipient strain that lacks the TRP1 region and that allows a high efficiency of gene deletion.

Deletion of the PAT1 Gene Affects Translation Initiation and Suppresses a PAB1 Gene Deletion in Yeast

ABSTRACT The yeast poly(A) binding protein Pab1p mediates the interactions between the 5′ cap structure and the 3′ poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. ThePAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

Cloning of a eukaryotic regulatory gene.

SummaryFrom a pool of hybrid plasmids carrying Sau3A fragments representing the entire yeast (S. cerevisiae) genome, a DNA fragment containing the regulatory gene PPR1 was cloned by complementation of a non-inducible ppr1 mutation which confers to the cells an increased sensitivity to 6-azauracil. Cells containing the cloned DNA regained the ability to induce the synthesis of URA1 and URA3 gene products controlled by PPR1. A physical map has been constructed and the study of subcloned restriction endonuclease fragments from the original yeast DNA fragment allowed us to localize the wild-type PPR1 regulatory gene within a 3 kilobase-pair region. The ppr1 RNA level was measured and the hybridization data indicate in a wild-type strain a low efficiency of transcription of PPR1 as compared to the structural URA3 gene, without effect of inducing conditions.

Mpe1, a zinc knuckle protein, is an essential component of yeast cleavage and polyadenylation factor required for the cleavage and polyadenylation of mRNA.

ABSTRACT In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene,MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice ofACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3′-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3′ end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3′ end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.

The duplicated Saccharomyces cerevisiae gene SSM1 encodes a eucaryotic homolog of the eubacterial and archaebacterial L1 ribosomal proteins.

A previously unknown Saccharomyces cerevisiae gene, SSM1a, was isolated by screening for high-copy-number suppressors of thermosensitive mutations in the RNA14 gene, which encodes a component from the polyadenylation complex. The SSM1 a gene codes for a 217-amino-acid protein, Ssm1p, which is significantly homologous to eubacterial and archaebacterial ribosomal proteins of the L1 family. Comparison of the Ssm1p amino acid sequence with that of eucaryotic polypeptides with unknown functions reveals that Ssm1p is the prototype of a new eucaryotic protein family. Biochemical analysis shows that Ssm1p is a structural protein that forms part of the largest 60S ribosomal subunit, which does not exist in a pool of free proteins. SSM1 a is duplicated. The second gene copy, SSM1b, is functional and codes for an identical and functionally interchangeable Ssm1p protein. In wild-type cells, SSM1b transcripts accumulate to twice the level of SSM1a transcripts, suggesting that SSM1b is responsible for the majority of the Ssm1p pool. Haploid cells lacking both SSM1 genes are inviable, demonstrating that, in contrast with its Escherichia coli homolog, Ssm1p is an essential ribosomal protein. Deletion of the most expressed SSM1b gene leads to a severe decrease in the level of SSM1 transcript, associated with a reduced growth rate. Polysome profile analysis suggests that the primary defect caused by the depletion in Ssm1p is at the level of translation initiation.