Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

钩端螺旋体liprmA基因的表达及其产物的纯化与功能分析

以钩端螺旋体基因组DNA为模板,通过酶联聚合反应（PCR）得到钩端螺旋体中prmA的同源基因liprmA的全基因编码序列,并克隆到原核表达载体pET22b中。通过优化大肠杆菌培养和诱导条件,含目的蛋白的融合蛋白可溶表达量达到40 mg/L,约占菌体总蛋白的40%。经Ni-NTA His Bind亲和柱纯化,得到纯度大于95%的目的蛋白。氨基酸序列同源性分析显示liPrmA与原核生物和真核生物的核糖体蛋白L11甲基化转移酶的功能域一级结构高度一致;活性分析显示,纯化的liPrmA有钩端螺旋体核糖体蛋白L11甲基化转移酶的活性。     

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.     

Nucleotide sequence of four genes encoding ribosomal proteins from the 'S10 and spectinomycin' operon equivalent region in the archaebacterium Halobacterium marismortui

Four genes encoding ribosomal proteins HmaS17, HmaL14, HmaL24 and HS3, have been identified in the lambda EMBL3 clone PP*7 from a genomic library of the archaebacterium Halobacterium marismortui. The clone contains genes from the 'S10 and spectinomycin' operon equivalent region. Three of the deduced proteins are homologous to the corresponding Escherichia coli and Methancoccus vannielii S17, L14 and L24 proteins, as well as to eukaryotic proteins from rat or yeast. HS3 was identified as an extra protein corresponding to the gene product for orfc in M. vannielii and the eukaryotic ribosomal protein RS4 from rat. The equivalence of HmaL24 (HL16) and E. coli L24, which share only 28% identical amino acid residues, could now be shown by localizing the HmaL24 gene at the same position in the cluster.     

Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster.

We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a lambda gt11 expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding S11) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation.     

Nucleotide sequences of Bacillus stearothermophilus ribosomal protein genes: part of the ribosomal S10 operon

Restriction fragments from Bacillus stearothermophilus chromosomal DNA were cross-hybridized with the Escherichia coli ribosomal protein L2 gene rplB. A 2-kb EcoRI fragment which showed cross-hybridization was cloned into the M13 phage and sequenced by the dideoxy chain-terminating method. Comparison of the deduced amino-acid sequences with the corresponding sequences of E. coli ribosomal proteins showed that this fragment contains the region encoding the C-terminus of L2, the genes encoding S19, L22, S3 as well as the N-terminus of L16. Thus the organization of this gene cluster is the same as that in the S10 operon of E. coli. The deduced sequences of proteins L22 and S3, which have not been determined so far, were found to have 52% or 55% amino-acid identity, respectively, with those of the corresponding proteins in E. coli. The deduced B. stearothermophilus S19 protein sequence was in accordance with the reinvestigated protein sequence (H. Hirano, personal communication).     

Ribosomal protein gene cluster of Halobacterium halobium: nucleotide sequence of the genes coding for S3 and L29 equivalent ribosomal proteins

A 1643 base pair fragment encoding the S3 and L29 equivalent ribosomal proteins has been sequenced from the archaebacterium Halobacterium halobium. The incomplete open reading frame present upstream from the S3 gene encodes a protein homologous to the eubacterial ribosomal protein L22. The initiation codons of the S3 and L29 genes overlap with the termination codons of the upstream genes. A tight physical organization suggests that these genes are transcribed as a polycistronic operon. Peculiarities of the protein structure and gene organization are discussed.