Conversion of a cosubstrate to an inhibitor: phosphorylation mutants of nicotinic acid phosphoribosyltransferase.

Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) forms nicotinic acid mononucleotide (NAMN) and PPi from 5-phosphoribosyl 1-pyrophosphate (PRPP) and nicotinic acid (NA). The Vmax NAMN synthesis activity of the Salmonella typhimurium enzyme is stimulated about 10-fold by ATP, which, when present, is hydrolyzed to ADP and Pi in 1:1 stoichiometry with NAMN formed. The overall NAPRTase reaction involves phosphorylation of a low-affinity form of the enzyme by ATP, followed by generation of a high-affinity form of the enzyme, which then binds substrates and produces NAMN. Hydrolysis of E-P then regenerates the low-affinity form of the enzyme with subsequent release of products. Our earlier studies [Gross, J., Rajavel, M., Segura, E., and Grubmeyer, C. (1996) Biochemistry 35, 3917-3924] have shown that His-219 becomes phosphorylated in the N1 (pi) position by ATP. Here, we have mutated His-219 to glutamate and asparagine and determined the properties of the purified mutant enzymes. The mutant NAPRTases fail to carry out ATPase, autophosphorylation, or ADP/ATP exchanges seen with wild-type (WT) enzyme. The mutants do catalyze the slow formation of NAMN in the absence of ATP with rates and KM values similar to those of WT. In striking contrast to WT, NAMN formation by the mutant enzymes is competitively inhibited by ATP. Thus, the NAMN synthesis reaction may occur at a site overlapping that for ATP. Previous studies suggest that the yeast NAPRTase does not catalyze NAMN synthesis in the absence of ATP. We have cloned, overexpressed, and purified the yeast enzyme and report its kinetic properties, which are similar to those of the bacterial enzyme.

Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I.

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.

RRN11 encodes the third subunit of the complex containing Rrn6p and Rrn7p that is essential for the initiation of rDNA transcription by yeast RNA polymerase I.

A new gene, RRN11, has been defined by certain rrn mutants of Saccharomyces cerevisiae which are defective specifically in the transcription of 35 S rRNA gene by RNA polymerase I (pol I). We have cloned the gene and found that it encodes a protein of 507 amino acids. We have used a strain with the chromosomal RRN11 deleted and carrying HA1 epitope-tagged RRN11 on a plasmid to isolate a protein complex containing the protein encoded by RRN11. This protein complex complemented rrn6 mutant extracts, which were previously shown to be deficient in the essential pol I transcription factor called Rrn6/7 complex or core factor (CF). The CF complex was previously shown to consist of three proteins, the 102- and 60-kDa subunits encoded by RRN6 and RRN7, respectively, and the 66-kDa subunit. The results of the above complementation experiments combined with mobility of Rrn11p in SDS-polyacrylamide gel electrophoresis analysis relative to Rrn6p and Rrn7p led to the conclusion that RRN11 encodes the 66-kDa subunit of CF. Glutathione S-transferase-Rrn11p fusion protein was found to bind strongly to 35S-labeled Rrn6p and Rrn7p but only weakly to 35S-labeled TATA-binding protein. Similarly, glutathione S-transferase-Rrn7p fusion protein bound strongly to 35S-labeled Rrn6p and Rrn11p but only weakly to 35S-labeled TATA-binding protein. These results are consistent with the fact that one can purify CF consisting of Rrn6p, Rrn7p, and Rrn11p from yeast cell extracts, but the purified complex does not contain TATA-binding protein. RRN11 was shown to be an essential gene, and [3H]uridine pulse experiments demonstrated directly that RRN11 is essential for rDNA transcription by pol I in vivo. Thus all three subunits of CF are essential for rDNA transcription. Because of the resemblance of CF to mammalian essential pol I transcription factor SL1, the amino acid sequences of Rrn11p and the other two subunits of CF were compared with those of the three TATA-binding protein-associated factors (TAFs) in the human SL1, TAFI48, TAFI63, and TAFI110. No significant similarity was detected between two sets of the proteins. Similarity as well as differences between CF and SL1 are discussed.

Organization of the centromeric region of chromosome XIV in Saccharomyces cerevisiae.

A 15.1 kb fragment of the yeast genome was allocated to the centromeric region of chromosome XIV by genetic mapping. It contained six bona fide genes, RPC34, FUN34, CIT1 (Suissa et al., 1984), RLP7, PET8 and MRP7 (Fearon and Mason, 1988) and two large open reading frames, DOM34 and TOM34. RPC34 and RLP7 define strictly essential functions, whereas CIT1, PET8 and MRP7 encode mitochondrial proteins. The PET8 product belongs to a family of mitochondrial carrier proteins. FUN34 encodes a putative transmembraneous protein that is non-essential as judged from the normal growth of the fun34-::LUK18(URA3) allele even on respirable substrates. TOM34 codes for a putative RNA binding protein, and DOM34 defines a hypothetical polypeptide of 35 kDa, with no significant homology to known proteins. The region under study also contains two divergently transcribed tDNAs, separated only by a chimeric transposable element. This tight tDNA linkage pattern is commonly encountered in yeast, and a general hypothesis is proposed for its emergence on the Saccharomyces cerevisiae genome. RPC34, RLP7, PET8 and MRP7 are unique on the yeast genome, but the remaining genes belong to an extant centromeric duplication between chromosome III and XIV.

Two distinct yeast proteins are related to the mammalian ribosomal polypeptide L7.

The RLP7 gene of Saccharomyces cerevisiae was cloned, sequenced and localized to the right arm of chromosome XIV, close to the centromere. It encodes a predicted polypeptide (RLP7p) of 322 amino acids, with a calculated molecular mass of 36 kDa and an isoelectric point of 9.6. Putative open reading frames very similar to RLP7 are present in two other yeasts, Kluyveromyces lactis and Candida utilis. The RLP7p gene product has significant sequence similarity to the S. cerevisiae YL8 polypeptide of the large ribosomal subunit (Mizuta et al., 1992), itself homologous to the L7 subunit of mammalian ribosomes. However, RLP7p and YL8 do not functionally replace each other, since an rlp7-delta::HIS3 strain is completely inviable. Judging from its predicted mass, isoelectric point and amino acid sequence, RLP7p does not correspond to any ribosomal component biochemically identified so far in S. cerevisiae, and also differs from all known ribosomal proteins by the low codon usage bias of its gene.

Interactions between three common subunits of yeast RNA polymerases I and III.

The AC40 and AC19 subunits (encoded by RPC40 and RPC19) are shared by yeast RNA polymerases I and III and have a local sequence similarity to prokaryotic alpha subunits. Mutational analysis of the corresponding "alpha motif" indicated that its integrity is essential on AC40 subunit but is not essential on AC19 subunit. By applying the two-hybrid method, these two polypeptides were shown to associate in vivo. Extragenic suppression of rpc19 and rpc40 mutations confirmed that AC19 and AC40 subunits interact with each other in vivo and revealed an interaction with ABC10 beta subunit [encoded by RPB10; Woychick, N. A. & Young, R.A. (1990) J. Biol. Chem. 265, 17816-17819], one of the five polypeptides common to all three nuclear RNA polymerases. A correction of the RPB10 sequence showed that ABC10 beta subunit is a 70-amino acid polypeptide, as confirmed by peptide microsequencing. These results suggest that the assembly of RNA polymerase I and III requires the association of ABC10 beta subunit with an AC19/AC40 heterodimer.

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.

Two yeast chromosomes are related by a fossil duplication of their centromeric regions.

A 15 kbp fragment of the Saccharomyces cerevisiae genome was cloned and localised to the centromeric region of chromosome XIV by genetic linkage and DNA sequencing. It had a strong sequence similarity and a conserved gene linkage and transcriptional orientation relatively to the centromeric region of chromosome III, indicating a fossil interchromosomal duplication of several linked genes. On chromosome XIV, the duplicated fragment included the centromere, four genes (FUN34, CIT1 and two tDNAs), one open reading frame (DOM34) and a truncated delta element. Additional inserts bearing unique genes were present on the centromeric region of chromosome III. The level of silent substitutions indicated a relatively ancient genetic separation, pre-dating the emergence of S. cerevisiae and S. douglasii as distinct species. The ensuing evolution of the duplicated regions retained strict sequence identity for the two tDNAs pairs, but was partially divergent for CIT1 and FUN34, and generated a probable pseudogenic equivalent of DOM34 on chromosome III. Extant multigenic duplications of this type might play an important role in the evolution of eukaryotic genomes.

Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes.

Polygalacturonase (Peh) and other pectolytic enzymes play a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have sequenced the peh gene of Erwinia carotovora subsp. carotovora, and identified its product as a precursor of molecular weight 42,639, and a mature protein of molecular weight 42,200. A putative KdgR-binding site was identified in the region 5' to the peh gene. The Peh protein showed significant homology with Peh from tomato. In addition, we have found homologies between pectin methylesterase and pectate lyase from Erwinia and their counterparts in tomato. These homologies are described, and their significance discussed.