Biomedical subjects

S Tapio

Publications and source records attributed to S Tapio.

7 recordsLinked to original sources

PubMed1992

Molecular characterization of the MalT-dependent periplasmic alpha-amylase of Escherichia coli encoded by malS.

malS, the gene encoding the periplasmic alpha-amylase, is under the regulatory control of the MalT protein, the gene activator of the Escherichia coli maltose system. We sequenced the DNA region encoding malS and its control elements. malS consists of an open reading frame of 2,028 base pairs encoding a protein of 676 amino acids with a deduced molecular weight of 75,664 including a typical amino-terminal signal sequence of 17 amino acids. The deduced amino acid sequence of malS was compared with that of other proteins. We found homologies to alpha-amylases and a cyclodextrin glucanotransferase but not to beta-amylases. In addition, MalS showed significant homology to another maltodextrin-utilizing and MalT-dependent enzyme of E. coli, maltodextrin glucosidase (MalZ) but not to amylomaltase (MalQ), the major maltodextrin-degrading enzyme. Conserved regions that have been proposed to constitute enzymatically active sites in alpha-amylases are present in MalS. Two of these sequences can also be found in the amino terminus of the lambda-receptor, a maltodextrin-specific channel in the outer membrane. The nucleotide sequence of the control region of malS revealed MalT binding sites correctly spaced for the start of the malS transcript. At the position 219 base pairs upstream of malS, we found a divergently transcribed gene, bax, which has been recognized previously. Downstream of malS, after a 513-base pair intergenic region, lies the convergently transcribed gene avtA, which codes for the alanine/valine transaminase.

Amino Acid Sequence↗

PubMed1991

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.

Alleles↗

PubMed1991

The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase.

We have characterized a maltodextrin glucosidase, previously described as a maltose-inducible, cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside in Escherichia coli. The gene encoding the enzyme activity, referred to as malZ, is located at 9.3 min on the chromosomal map. We cloned the gene in a high copy number vector and purified the enzyme. It is a monomer, with an apparent molecular weight of 65,000. The enzyme degrades maltodextrins, ranging from maltotriose to maltoheptaose, to shorter oligosaccharides, the final hydrolysis products being maltose and glucose. We measured the kinetic parameters, Km and Vmax, for the hydrolysis to glucose of the five different substrates. The binding of the substrate is enhanced by increasing the number of glucosyl residues in the maltodextrin. In contrast, the maximum rate of hydrolysis (Vmax) is fastest for maltotriose. To study the mode of action of the enzyme, we quantitatively measured the amount of free glucose liberated from the different maltodextrin substrates after a long incubation. More glucose is liberated from the long dextrins, as compared to the shorter ones, showing that the primary hydrolysis product was glucose, not maltose. Furthermore, [14C]maltotriose, specifically labeled at the reducing end, was hydrolyzed to [14C]glucose and unlabeled maltose. These data demonstrate that the malZ gene product is a maltodextrin glucosidase, liberating glucose from the reducing end of malto-oligosaccharides. The nucleotide sequence of malZ and the deduced amino acid sequence showed that malZ encodes a protein with a molecular weight of 68,960. Homology to glucosidases, alpha-amylases, and pullulanases were observed. Conserved regions thought to represent active sites in dextrin hydrolases were found in the MalZ protein.

Amino Acid Sequence↗

PubMed1990

Antisuppression by mutations in elongation factor Tu.

Two slow-growing kirromycin-resistant Escherichia coli mutants with altered EF-Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene. Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here. Ap, the tufA gene product of strain D2216 (from A. Parmeggiani), has previously been shown to give an increased GTPase activity. The slow cellular growth rates of both EF-Tu mutants are correlated with decreased translational elongation rates. Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs [tyrT(Su3), trpT(Su9), glyT(SuAGA/G)], but have only little or no effect on misreading by wild-type tRNAs. A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF-Tu mutations. The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double-mutant strain has an elevated temperature optimum (42 degrees C) for growth rate, translation rate and nonsense suppression. Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.

Amino Acids↗

PubMed1990

Impaired in vitro kinetics of EF-Tu mutant Aa.

The kirromycin-resistant EF-Tu mutant Aa, previously shown to be an antisuppressor for nonsense and missense suppressor tRNAs, has been characterised in a poly(U)-primed translation system in vitro. Two major defects were found in the function of the mutant. First, the dissociation constant for Aa binding to Phe-tRNA(Phe) was increased tenfold compared to wild-type EF-Tu. Second, kcat/Km for the interaction between the EF-Tu.GTP.aa-tRNA complex and the ribosome was decreased by the mutation to one third of its wild-type value. No differences were observed between mutant and wild-type factor in the regeneration of EF-Tu.GTP from EF-Tu.GDP via EF-Ts or in the mistranslation frequency by Leu-tRNA(4Leu). The relation between the in vitro results and the mutant phenotype in vivo is discussed.

Bacterial Proteins↗

PubMed1988

Antagonistic effects of mutant elongation factor Tu and ribosomal protein S12 on control of translational accuracy, suppression and cellular growth.

Kirromycin-resistant mutant forms of elongation factor Tu, which are coded by tufA (Ar) or tufB (Bo) and are associated with an increased rate of translational error formation, have been analysed. In vivo, Ar was found to increase misreading as well as suppression of non-sense codons irrespective of Bo in a strain with wild type ribosomes. It is therefore not necessary to evoke both tufA (Ar) and tufB (Bo) mutations together in order to increase translational error as suggested earlier [1]. When combined with a hyperaccurate ribosomal rpsL (S12) mutation, Ar counteracts the restrictive effects on translational error formation caused by the altered protein S12, thus restoring the levels of missense error in vitro and non-sense error and suppression in vivo to near wild type values. As judged from in vitro experiments this results principally from a lowered selectivity of the Ar ternary complex at the initial discrimination step on the ribosome during translation. In vivo, this compensatory effect on the rpsL mutation on non-sense error formation and suppression is seen irrespective of the nature of tRNA or codon context. Furthermore, the tufA mutation enhances the cellular growth rate of the rpsL mutant, whereas it decreases growth of strains with normal ribosomes. Inactivation of one of the two genes coding for EF-Tu (tufB), while leaving the other gene (tufA) intact, can by itself, increase non-sense error formation and suppression.

Codon↗

PubMed1986

Mutant EF-Tu increases missense error in vitro.

We have studied the consequences of mutational alteration in the structure of EF-Tu on the missense errors and proofreading activity of bacterial ribosomes in vitro. Our data show that the EF-Tu Bo mutant form of EF-Tu (van der Meide et al. 1983a) is inactive in polypeptide synthesis on the ribosome, even though it binds aminoacyl-tRNA. A second mutant form, EF-Tu Ar (van der Meide et al. 1983a), is active in polypeptide synthesis but supports a much higher messense incorporation with either leucine isoacceptor 2 or leucine isoacceptor 4 in the in vitro system. Further analysis of the kinetic basis of this enhanced missense frequency revealed that the mutation responsible for the alteration in EF-Tu Ar increases the errors at both the proofreading step and the initial selection. In this respect the effect of this particular mutation is similar to the mode of action of the antibiotic kanamycin (Jelenc and Kurland 1984).

Escherichia coli↗