Regulation of Expression of Elongation Factor EF-Tu 

The expression of bacterial proteins is mainly regulated at the level of transcription. It is, therefore, highly dependent on the function of promoters. The present study is a part of a broader project to learn how to deduce, in Gram positive bacteria B. stearothermophilus and B. subtilis, the level of expression of genes from the primary structure of their regulatory regions, e.g. promoters, of their operons. 

The complete primary structure of the str operon (Fig. 1, in green color) harboring the tuf gene, coding for EF-Tu of B. stearothermophilus, was determined. The operon was found to consist of the following genes: ybxF (unknown function), rpsL and rpsG, coding for ribosomal proteins S12 and S7, respectively, fus and tuf, coding for elongation factor EF-G and elongation factor EF-Tu, respectively.

Fig. 1: Schema of the str operon of Bacillus stearothermophilus

Two promoters were identified to direct transcription of the operon: the main operon promoter strp, situated upstream of the ybxF and directing synthesis of the polycistronic mRNA, and tufp, situated in the fus–tuf intergenic region and directing synthesis of the tuf mRNA. Deletion mapping of the two promoters using CAT assays revealed the existence of oppositely acting cis elements located upstream (5') of the promoters providing the tufp with about 10 times stronger activity in comparison to strp (Fig. 2).

Fig. 2: Comparison of the transcription activity of strp and tufp of Bacillus stearothermophilus

This fully correlated with the concentration difference between the strp directed polycistronic mRNA and the tufp–directed transcripts. Similar results were obtained with B. subtilis. These data imply that in Gram-positive Bacilli, the natural, about ten times higher level of EF-Tu over e.g. EF-G in the cell, is primarily the result of differential transcription and different promoter strength, explaining for the first time the abundance of EF-Tu over EF-G. This is a new strategy, clearly dissimilar from what has been found in Gram-negative bacteria E. coli till now.

At present our effort has been aimed at the S10 ribosomal protein operon, which is situated immediately downstream of the str operon (Fig 3).

Fig. 3: Schema of the S10 operon of Bacillus stearothermophilus

The experiments are in progress to determine the transcriptional rules in this operon dependent on the activity of two tandem promoters located immediately upstream of the first gene, and to compare their strength and regulation with those of tufp and strp. 

As described above, the ybxF gene was found to extend the str operon in both B. stearothermophilus and B. subtilis upstream of the highly conserved rpsL, rpsG, fus and tuf genes. This is a new observation. ybxF has not been detected in Gram-negative bacteria and its significance in Gram positive Bacilli has been investigated.

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