Potencial eólico

The gene encoding NusB was first identified by genetic analysis o f E. coli

mutants that block transcriptional antitermination by the N protein of bacteriophage X

(Friedman et a l, 1976). The importance o f this gene in antitermination was confirmed by experiments showing that premature transcription termination takes place in the rRNA opérons of nusB mutants (Sharrock et al, 1985) . The product of the nusB gene,

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Figure 4.9.- Purification of the in vivo expressed 6xHis tagged putative NusB protein of

M. leprae. The figure shows the electrophoretic separation of the fractions obtained by purification/refolding in the Ni-NTA resin. Lane 1: molecular weight markers; lane 2: total protein from cells induced by 2mM IPTG for 3 hrs, showing the overexpression of putative recombinant NusB protein; lanes 3 and 4: proteins released from the column in the first (lane 3) and last (lane 4) washing steps; lanes 5 to 6; proteins eluted from the Ni-NTA column with 250 mM Imidazole.

a protein o f -14 KDa (NusB) that has been shown to form part of the transcription complex, seems to be essential for efficient chain elongation during rRNA synthesis.

In our efforts to understand the role played by transcription antitermination in the regulation o f rRNA synthesis in mycobacteria, we have been looking at homologues of the E. coli factors that have been described to be involved in antitermination in rrn

opérons. Since one of the most relevant features o f the E. coli system is the interaction between ribosomal protein SIO, the NusB protein and boxA RNA, we decided to identify, clone and characterise the mycobacterial equivalent to E. coli NusB.

In this chapter we have described a family of proteins related to E. coli NusB proteins, that share a “domain” of 82 aminoacids of relatively high homology, as well as a short arginine rich sequence in the amino terminus. Among these proteins there are M

leprae ecuà M.tuberculosis homologues. It seems highly likely that proteins belonging to this family fulfil similar functions, since at least half of their aminoacid sequence is similar. However, proteins with homology to E. coli NusB are not identical. There are important differences between them in size, charge, and isolectric point that are likely to affect the function of the protein. One of the main difficulties that we have found in studying NusB is our limited knowledge of this transcription factor: NusB protein has not been studied in any species other than E. coli, and even in this bacterium the information that we have is not extensive. We do not know, for instance, what is the significance in terms of antitermination function o f the homologies and differences in the aminoacid sequences that we have found. Recently, it has been proposed that a short region of the E. coli NusB (1-10 aa) containing an arginine-rich RNA-binding motif (ARM) similar to the one found in other RNA-binding proteins like BIV tat and Rev protein, could be responsible for the RNA binding properties of NusB (Altieri et al., 1997). The putative mycobacterial NusB protein does not seem to have a ARM domain.

Especially intriguing are the differences between M. leprae and M.tuberculosis

putative NusB proteins. These two species of pathogenic mycobacteria are known to be closely related. Indeed, the putative NusB proteins o f these two species share an almost identical domain, as judge by the aminoacid sequence homology. However, they differ markedly in size, since the M. leprae NusB homologue has 32 additional aminoacids at

protein than the M. leprae homologue. The functional significance of the differences between proteins of closely related species is difficult to estimate. If the only role of NusB is to interact with ribosomal protein SIO and boxA RNA to produce antitermination of transcription, the variability of the protein between different species should be limited by boxA and SIO. NusB can be very different between species, but the differences should not change its ability to interact with NusB and boxA. The ribosomal protein SIO of M leprae and M.tuberculosis is highly conserved (only 5 out of 102 differences in the aa sequence) and boxA sequences are identical in all the mycobacterial species. Why is the putative NusB protein different? Is there any evolutionary advantage in having such a different NusB protein? One possible explanation is that the conserved domain is sufficient for the binding function of putative NusB proteins, and that the extra aa do not alter such capacity. Another possibility is that the extra aminoacids fulfil other roles in the bacterial cell, although we have not found any protein in the databank with homology to the non-conserved aminoacids of the putative NusB protein o f M. leprae. It is also possible that the extra aminoacids in the M. leprae NusB homologue, by affecting the secondary structure of the protein, will change its capacity to bind ribosomal protein SIO and boxA RNA. In subsequent chapters we will demonstrate that there are major differences between the binding properties of the mycobacterial NusB and those reported for the E. coli protein.