The ribosomal protein S8 from Thermus thermophilus VK1

Abstract

The gene of the ribosomal protein S8 from Thermus thermophilus VK1 has been isolated from a genomic library by hybridization of an oligonucleotide coding for the N-terminal amino acid sequence of the protein, amplified by PCR and sequenced. Nucleotide sequence reveals an open reading frame coding for a protein of 138 amino acid residues (Mr 15 839). The codon usage shows that 94% of the codons possess G or C in the third position, and agrees with the preferential usage of codons of high G+C content in the bacteria of the genus Thermus. The amino acid sequence of the protein shows 48% identity with the protein from Escherichia coli. Ribosomal protein S8 from 7: thermophilus has been expressed in E. coli under the control of the T7 promoter and purified to homogeneity by heat treatment of the extract followed by cation-exchange chromatography. Conditions were defined in which 7: thermophilus protein S8 binds specifically an homologous 16s rRNA fragment containing the putative S8 binding site with an apparent association constant of 5x10' M-'. The overexpressed protein binds the rRNA with the same affinity as that extracted from 7: thermophilus, indicating that the thermophilic protein is correctly folded in E. coli. The specificity of this binding is dependent on the ionic strength. The protein S8 from 7: thermophilus recognizes the E. coli rRNA binding site as efficiently as the S8 protein from E. coli. This result agrees with sequence comparisons of the S8 binding site on the small subunit rRNA from E. coli and from 7: thermophilus, showing strong similarities in the regions involved in the interaction. It suggests that the structural features responsible for the recognition are conserved in the mesophilic and thermophilic eubacteria, despite structural pecularities in the thermophilic partners conferring thermostability.Although crystals of ribosomes as well as of ribosomal particles diffracting at correct resolution have been obtained (see e.g. Yonath et al., 1990;Yusupov et al., 1991), determination at atomic level of the structure of such multimolecular ribonucleoprotein complexes is obviously a long-term pro-

Ribosomal Protein S15 from Thermus Thermophilus

“…As overexpression of some thermophilic ribosomal protein genes has been shown to be toxic for E. coli cells (Ramakrishnan and Gerchman, 1991;Vysotskaya et al, 1994), 7: thermophilus S15 overproduction was obtained using the Studier's host vector system utilised successfully for expression of toxic genes in E. coli (Studier et al, 1990). This system contains the strain Ntermophilus.…”

“…All 7: thermophilus S15 was found in the soluble fraction. Therefore, it can be purified avoiding urea treatment, under non-denaturing conditions, using the standard technique for purification of 7: thermophilus ribosomal proteins from overproducing strains (Vysotskaya et al, 1994;Nikonov et al, 1996; Materials and Methods) with the following modifications. The MgCI, concentration was increased to 150 mM in the lysis buffer and the heating was restricted to 55 "C for 10 min in the presence of 0.8 M NaCI.…”

Preliminary NMR studies of Thermus thermophilus ribosomal protein S19 overproduced in Escherichia coli

“…Plasmid pBR322‐tthS8 [16]for initial gene sequencing was kindly provided by Dr. V. Vysotskaya. The ribosomal protein S19 was cloned into pACA vector using the `sticky feet'‐directed mutagenesis protocol [17, 18].…”

“…The purification procedure for the ribosomal protein S19 was almost the same as for the other recently overexpressed ribosomal proteins from T. thermophilus [16, 17]with some modifications. Briefly, 3 g of wet cells were ground with alumina and resuspended in 100 mM Tris‐HCl, pH 8.1, with 800 mM NaCl, 150 mM MgCl 2 , 5 mM β‐mercaptoethanol and DNase (0.5 mg/ml final concentration).…”

Ribosomal protein L22 from Thermus thermophilus: sequencing overexpression and crystallisation

“…Plasmid pBR322-tthS8 [21] was used in sequencing studies. This plasmid contained the spc and SIO operons of Z thermophilus (V. Vysotskaya, unpublished data).…”