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D K Jemiolo

Publications and source records attributed to D K Jemiolo.

10 recordsLinked to original sources
PubMed1995

UGA suppression by a mutant RNA of the large ribosomal subunit.

A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.

Base Sequence↗
PubMed1991

Mutations in 16S rRNA in Escherichia coli at methyl-modified sites: G966, C967, and G1207.

Mutations were constructed at three sites in 16S rRNA in E. coli by oligonucleotide-directed mutagenesis, and cloned into the rrnB operon on either pKK3535 or pNO2680. The mutated bases, G966, C967, and G1207, are located in the 3' major domain of 16S rRNA and are sites post-transcriptionally modified by methylation. We constructed a deletion mutation at C967 (delta 967) and three substitution mutations at each of the following sites: G966, C967, and G1207. By maxicell analysis, we found that all of the mutations were processed normally and incorporated into 30S subunits and 70S ribosomes. We found that delta 967 was a dominant lethal mutation while the substitution mutations at G966 and C967 had no effects on cell growth rate. The mutants C1207 and U1207 were shown to have dominant lethal phenotypes while A1207 had no effect on cell growth rate. These results help to establish the importance of methyl-modified regions to ribosome function.

Base Sequence↗
PubMed1986

Characterization of a collection of deletion mutants at the 3'-end of 16S ribosomal RNA of Escherichia coli.

Deletions were constructed in plasmid pKK3535 in the coding region for the 3'-end of E. coli 16S rRNA. The plasmid was cleaved with restriction endonuclease Hae2 under conditions favoring the production of single cut linear plasmid DNA and deletions were produced by digestion with exonuclease Bal31. Seven different deletions were isolated ranging in size from 90 to about 200 base pairs. Transcription of ribosomal DNA, processing of ribosomal RNA and incorporation of mutant rRNA into mutant particles was studied in UV-sensitive cells using a modified maxicell labeling procedure. The different mutants were missing defined features in the secondary structure of 16S rRNA and were characterized according to their stability, ability to be processed, sensitivity to colicin E3, and ability to bind ribosomal protein S1 and to interact with 50S subunits. These analyses show that the small stem and loop structure at positions 1350 to 1372 is necessary for the stability of rRNA. The deletion of the long terminal stem structure (1409-1491) in all mutant rRNAs does not block processing of the mutant rRNAs or S1 binding, although processing of the mutant rRNAs or S1 binding, although it does prevent the association of particles containing the mutant rRNA with 50S subunits.

Base Sequence↗
PubMed1985

Point mutations in the 3' minor domain of 16S rRNA of E.coli.

Point mutations were produced near the 3' end of E. coli 16S rRNA by bisulfite mutagenesis in a 121 base loop-out (1385 to 1505) in a heteroduplex of wild type (pKK3535) and deletion mutant plasmids. Two highly conserved, single stranded regions flank an irregular helix (1409-1491) in the area studied. Only a single mutation was isolated in the flanking regions, a transition at C1402, (normally methylated on the base and ribose in rRNA). Mutations occurred throughout the irregular helix. All mutant rRNAs were processed and assembled into 30S subunits capable of interacting with 50S subunits. Growth rates ranged from faster to significantly slower than cells with the wild type transcript. In particular, mutations at C1467 or C1469 cause slow growth. These two transitions (in a bulge region within the helix) reduced the bulge by additional base pairing.

Base Sequence↗
PubMed1985

A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro.

We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene. The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript. Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation. Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme.

Autoradiography↗
PubMed1982

Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin.

Tubulin was first treated with alkaline phosphatase-agarose to vacate the exchangeable nucleotide binding site and then tested for manganese binding sites by Mn(II) EPR. Buttlaire et al. ((1980) J. Biol. Chem. 255, 2164-2168) have shown that high affinity manganese binding occurs at a single site normally occupied by magnesium. We report that the number of high affinity manganese binding sites per mol of tubulin depends on the number of occupied exchangeable nucleotide binding sites. Thus, removal of nucleotides results in a loss of high affinity manganese binding sites. The EPR spectra of manganese bound to tubulin and to GTP are found to be qualitatively similar. These data indicate that high affinity manganese binding is the result of the formation of a metal-nucleotide complex at the exchangeable nucleotide binding site. In addition it was found that zinc, cobalt, and magnesium bind with approximately equal affinity to this site whereas calcium binds only weakly.

Alkaline Phosphatase↗
PubMed1982

Strongylocentrotus purpuratus spindle tubulin. II. Characteristics of its sensitivity to Ca++ and the effects of calmodulin isolated from bovine brain and S. purpuratus eggs.

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus and purified through cycles of temperature-dependent assembly and disassembly. At 37 degrees C, the majority of the cycle-purified spindle tubulin polymer is insensitive to free Ca++ at concentrations below 0.4 mM, requiring free Ca++ concentrations greater than 1 mM for complete depolymerization. However, free Ca++ at concentrations above 1 microM inhibits initiation of polymer formation without significantly inhibiting the rate of elongation onto existing polymer. At 15 degrees C and 18 degrees C, temperatures that are physiological for S. purpuratus embryos, spindle tubulin polymer is sensitive to free Ca++ at micromolar concentrations such that 3-20 microM free Ca++ causes complete depolymerization. Calmodulin purified from either bovine brain or S. purpuratus eggs does not affect the Ca++ sensitivity of the spindle tubulin at 37 degrees C, although both increase the Ca++ sensitivity of cycle-purified bovine brain tubulin. These results indicate that cycle-purified spindle tubulin and cycle-purified bovine brain tubulin differ significantly in their responses to calmodulin and in their Ca++ sensitivities at their physiological temperatures. They also suggest that, in vivo, spindle tubulin may be regulated by physiological levels of intracellular Ca++ in the absence of Ca++-sensitizing factors.

Animals↗
PubMed1980

Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate.

Calmodulin has been purified to homogeneity using an improved procedure that allows rapid processing of several kilograms of bovine brain. A calcium-dependent change in the electrophoretic mobility of calmodulin in the presence of sodium dodecyl sulfate (SDS) has been observed. Freshly prepared calmodulin or lyophilized calmodulin, stored at --80 degrees C for 1--7 months, migrates as a single band with an apparent molecular weight of 21 000 when the sample, gel and running buffer are made 0.1 mM in EDTA. When 0.1 mM CaCl2 is substituted for EDTA, freshly isolated calmodulin migrates as a single band with an apparent molecular weight of 15 000. More slowly migrating bands, in addition to the 15 000 molecular weight band, are observed when the stored protein is electrophoresed under the same conditions. Calcium binding experiments show that freshly prepared calmodulin binds 4 mol of calcium per mol of protein in the presence of 0.1% SDS in 0.1 mM CaCl2. Skeletal muscle troponin C, carp parvalbumin, and bovine brain S-100b do not show this mobility change. The calcium-dependent mobility change can be used to identify calmodulin in crude protein preparations. Calmodulin has been identified in the sperm of the sea urchin, Strongylocentrotus purpuratus, and purified. The urchin calmodulin activates cyclic nucleotide phosphodiesterase to the same extent as does brain calmodulin. We used several criteria to determine that calmodulin is not present as a soluble protein in Escherichia coli.

Animals↗