Structure and expression of elongation factor Tu from Bacillus stearothermophilus.
The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stearothermophilus was cloned and sequenced. This gene maps in the same context as the tufA gene of Escherichia coli str operon. Northern-blot analysis and primer extension experiments revealed that the transcription of the tuf gene is driven from two promoter regions. One of these is responsible for producing a 4.9-kb transcript containing all the genes of B. stearothermophilus str operon and the other, identified adjacent to the stop codon of the fus gene and designated tufp, for producing a 1.3-kb transcript of the tuf gene only. In contrast to the situation in E. coli, the ratio between the transcription products was found to be about 10:1 in favour of the tuf gene transcript. This high transcription activity from the tufp promoter might be accounted for by the presence of an extremely A+T-rich block consisting of 29 nucleotides which immediately precedes the consensus -35 region of the promoter. A very similar tuf gene transcription strategy and the same tufp promoter organization with the identical A/T block were found in Bacillus subtilis. The tuf gene specifies a protein of 395 amino acid residues with a molecular mass of 43,290 Da, including the N-terminal methionine. 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 376 and a low affinity for kirromycin determined by zone-interference gel electrophoresis (Kd approximately 8 microM) and by polyacrylamide gel electrophoresis under non-denaturing conditions are in agreement with the reported resistance of this EF-Tu to the antibiotic. The replacement of the highly conserved Leu211 by Met was identified as a possible cause of pulvomycin resistance.
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo n.2, Praha 6, 166 37, Czech Republic.
L
L
Krásný
J R
JR
Mesters
L N
LN
Tieleman
B
B
Kraal
V
V
Fucík
R
R
Hilgenfeld
J
J
Jonák
D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D004269:DNA, Bacterial; D004352:Drug Resistance, Microbial; D001411:Geobacillus stearothermophilus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011728:Pyridones / Q000494:pharmacology; D016415:Sequence Alignment
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D011728:Pyridones; C012521:pulvomycin; D010444:Peptide Elongation Factor Tu; C008536:mocimycin