Rates of assembly and degradation of bacterial ice nuclei

The kinetics of ice-nucleus assembly from newly synthesized nucleation protein were observed following induction of nucleation gene expression in the heterologous host Escherichia coli. Assembly was significantly slower for the small proportion of ice nuclei active above -4.4 degrees C; this was consistent with the belief that these nuclei comprise the largest aggregates of nucleation protein. The kinetics of nucleus degradation were followed after inhibiting protein synthesis. Nucleation activity and protein showed a concerted decay, indicating that most of the functional ice nuclei are in equilibrium with a single cellular pool of nucleation protein. A minority of the ice nuclei decayed much more slowly than the majority; presumably their nucleation protein was distinct either by virtue of different structure or different subcellular compartmentalization, or because of its presence in a metabolically distinct subpopulation of cells.     

Antibodies to pollen exines

A polyclonal antiserum and monoclonal antibodies have been prepared to purified pollen exines of Calocedrus decurrens Florin. The location of the antigen is in the exine, as shown by light-and electron-microscopic immunocytochemistry. The greatest reduction in antibody binding follows treatment of the exine with chemicals known to alter sporopollenin. These results provide evidence that sporopollenin is antigenic. Exines of ten species of gymnosperms and angiosperms also bound the polyclonal antiserum, indicating similarity of sporopollenin structure.     

Dwarf mutants ofBrassica: Responses to applied gibberellins and gibberellin content

Eight rapid-cyclingBrassica genotypes differing in height were treated with gibberellins (GAs) by syringe application to the shoot tip. The height of two genotypes ofBrassica napus, Bn5-2 and Bn5-8, andB. rapa mutants,dwarf 1 (dwf1) anddwarf 2 (dwf2), was unaffected by exogenous GA₃ at dosages up to 0.1 g/plant, a level which increased shoot elongation of normal genotypes. Thus, these dwarf mutants are GA-insensitive. In contrast to theB. napus dwarfs, twoB. rapa mutants,rosette (ros), anddormant (dor), elongated following GA₃ application. The dwarfros was most sensitive, responding to applications as low as 1 ng GA₃/plant. Furthermore,ros also responded to GA₁ and some of its precursors with decreasing efficacy: GA₃>ent-kaurenoic acid GA₁>GA₂₀GA₁₉=GA₄₄GA₅₃. Endogenous GAs were measured by gas chromatography-selected ion monitoring using [²H₂]GA internal standards for calibration, from shoots of the GA-insensitive genotypes Bn5-2, Bn5-8 which contained theB. napus mutantdwarf 1, and from a normal genotype Bn5-1. Concentrations of GA₁ and GA₂₀ averaged 3.2- and 4.6-fold higher, respectively, and GA₁₉ levels also tended to be higher in the dwarfs than in the normal genotype.     

NMR assignment of a KlbA intein precursor from Methanococcus jannaschii

The backbone and side chain resonance assignments of a precursor of the KlbA intein from Methanococcus jannaschii have been determined, based on triple-resonance experiments with the uniformly [¹³C,¹⁵N]-labeled protein.     

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.     

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding

Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.     

Protein splicing of the Deinococcus radiodurans strain R1 Snf2 intein

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues.     

Demonstration of the role of the DnaK chaperone system in assembly of 30S ribosomal subunits using a purified in vitro system

Recently, there has been controversy regarding the ability of the DnaK chaperone system to facilitate Escherichia coli 30S subunit assembly at otherwise nonpermissive conditions. Here, we present additional data indicating that purified DnaK chaperone assembled 30S subunits are functional. Additionally, explanations for reported differences are discussed.     

Control of protein splicing by intein fragment reassembly.

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.