The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.     

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.     

The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12

Penicillin-binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase-transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1B, was also determined. Penicillin-binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C beta-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1A and 1B and the other penicillin-binding proteins and beta-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1A, 1B and 3, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The other region was 19-40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.