A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

A Bacillus subtilis mutant spnA95 was isolated as resistant at 30 degrees C to the protein kinase C (PKC) inhibitor, sphinganine, and temperature sensitive for growth. As deduced by flow cytometry measurements, the mutant has a 35% reduced initiation mass at permissive temperature, resulting in initiation of DNA replication much earlier in the cell cycle than in the wild type. This modification is accompanied by a change in cell size, as determined by phase-contrast microscopy and flow cytometry. Therefore, this strain displays the characteristics of a novel cell clock mutant. spnA is a newly identified gene in B.subtilis and was shown to encode a cysteinyl-tRNA synthetase. At non-permissive temperature, the mutant was defective in the synthesis of P70, a protein with several characteristics of PKC (a cysteine-rich protein). As one possibility, we propose that the altered timing of replication may be due to the reduced synthesis of specific cysteine-rich proteins normally involved in controlling chromosomal replication initiation in B. subtilis.     

Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.     

Reconstitution and transphosphorylation of TGF-beta receptor complexes.

Transforming growth factor-beta (TGF-beta) signals by contacting two distantly related transmembrane serine/threonine kinases called receptors I (T beta R-I) and II (T beta R-II). TGF-beta binds to T beta R-II, which is a constitutively active kinase and this complex recruits T beta R-I, causing its phosphorylation and signal propagation to downstream substrates. The biochemical properties of this interaction were analyzed with reconstituted receptor systems. T beta R-I and T beta R-II baculovirally expressed at high levels in insect cells have the ligand binding properties of receptors expressed in mammalian cells, and form a complex in which T beta R-I phosphorylation is dependent on the kinase activity of T beta R-II. Furthermore, T beta R-I and T beta R-II can form a complex in vitro, and their cytoplasmic domains can specifically interact in a yeast two-hybrid system. In vitro complex formation with catalytically active T beta R-II is necessary and sufficient for T beta R-I phosphorylation, which within this complex does not require the catalytic activity of T beta R-I, thus mimicking T beta R-I phosphorylation in intact cells. In addition, T beta R-I phosphorylated in vitro remains associated with T beta R-II. These results suggest that T beta R-I and T beta R-II have affinity for each other, however, the ligand is required for stable complex formation under physiological conditions. Once formed, this complex is sufficient for T beta R-I phosphorylation by T beta R-II.     

The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence.

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.