ABSTRACT
The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified. The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies. The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2. 5 A spacing. The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 A spacing using synchrotron radiation.
Subject(s)
Bacterial Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Geobacillus stearothermophilus/genetics , Hydrolysis , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Transcription Factors/genetics , Transcription Factors/isolation & purificationABSTRACT
Spore formation is an extreme response of some bacteria to adversity. In Bacillus subtilis the proteins of the sin, sporulation inhibition, region form a component of an elaborate molecular circuitry that regulates the commitment to sporulation. SinR is a tetrameric repressor protein that binds to the promoters of genes essential for entry into sporulation and prevents their transcription. This repression is overcome through the activity of SinI, which disrupts the SinR tetramer through the formation of a SinI-SinR heterodimer. The interactions governing this curious quaternary transition are revealed in the crystal structure of the SinI-SinR complex. The most striking, and unexpected, finding is that the tertiary structure of the DNA-binding domain of SinR is identical with that of the corresponding domains of the repressor proteins, CI and Cro, of bacteriophage 434 that regulate lysis/lysogeny. This structural similarity greatly exceeds that between SinR and any bacterial protein or between the 434 repressor proteins and their homologues in the closely related bacteriophage lambda. The close evolutionary relationship implied by the structures of SinR and the 434 repressors provokes both comparison of their functions and a speculative consideration of the intriguing possibility of an evolutionary link between the two adaptive responses, sporulation and prophage induction.
Subject(s)
Bacillus subtilis/physiology , Bacillus subtilis/virology , Spores, Bacterial/genetics , Spores, Bacterial/physiology , Virus Activation/genetics , Amino Acid Sequence , Bacillus Phages/genetics , Bacillus Phages/physiology , Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Biological Evolution , Models, Molecular , Molecular Sequence Data , Protein Conformation , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/physiology , Sequence Homology, Amino Acid , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/physiologyABSTRACT
Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate. The extracellular ribonuclease from Bacillus intermedius (binase, RNase Bi) shares a common mechanism for RNA hydrolysis with mammalian RNases. Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage. Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GpC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied. The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.
