STRUCTURE AND EXPRESSION OF ELONGATION-FACTOR TU FROM BACILLUS-STEAROTHERMOPHILUS

The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stear othermophilus was cloned and sequenced. This gene maps in the same con text as the tufA gene of Escherichia coli sir operon. Northern-blot an alysis and primer extension experiments revealed that the transcriptio n of the tuf gene is driven fi om two promoter regions. One of these i s responsible for producing a 4.9-kb transcript containing all the gen es of B. stearothermophilus str operon and the other, identified adjac ent to the stop codon of the fits gene and designated tufp, for produc ing a 1.3-kb transcript of the tuf gene only. In contrast to the situa tion in E. coli, the ratio between the transcription products was foun d to be about 10:1 in favour of the tuf gene transcript. This high tra nscription activity from the tufp promoter might be accounted for by t he presence of an extremely A + T-rich block consisting of 29 nucleoti des which immediately precedes the consensus -35 region of the promote r. A very similar tuf gene transcription strategy and the same tufp pr omoter organization with the identical A/T block were found in Bacillu s subtilis. The tuf gene specifies a protein of 395 amino acid residue s with a molecular mass of 43,290 Da, including the N-terminal methion ine. A computer-generated three-dimensional homology model shows that all the structural elements essential for binding guanine nucleotides and aminoacyl-tRNA are conserved. The presence of serine at position 3 76 and a low affinity for kirromycin determined by zone-interference g el electrophoresis (K-d similar to 8 mu M) and by polyacrylamide gel e lectrophoresis under non-denaturing conditions are in agreement with t he reported resistance of this EF-Tu to the antibiotic. The replacemen t of the highly conserved Leu211 by Met was identified as a possible c ause of pulvomycin resistance. (C) 1998 Academic Press.