INVOLVED m
ANTITERMINATION.
RNA sequences play an essential role in controlling the termination of transcription. Since many prokaryotic terminators require a hairpin to be formed in the RNA, transcription termination can be regulated by changing the conformation of the RNA, a phenomenon known as attenuation of transcription (see for example Keller and Calvo, 1979 or Yanofsky et al., 1996). Another way of controlling transcription termination is by altering the transcription complex to a form with decreased termination efficiency. The best characterized example of this kind of control is the N-mediated antitermination in bacteriophage X, the system in which transcription antitermination was first described (for a review see Friedman and Court, 1995). N-mediated transcription antitermination requires cis-acting sequences termed nut (Y-wrilization) sites: a single nut site is sufficient to cause antitermination at terminators located several kilobases downstream. A X nut
site consists of two sequence elements: a conserved boxA sequence ((CAJ)GCUCUU(U/-)A) (Friedman and Olson, 1983) and an inverted repeat sequence that forms a stem-loop structure in the RNA (boxB) (Rosemberg et a l, 1978). Sequence elements related to boxA are also found in bacterial opérons, while boxB is specific to the phage genome. boxB is responsible for the specificity of the interaction of N with the nut site (Lazinski et a l, 1989); boxA is involved in the binding of the host proteins (see for example Friedman and Olson, 1983).
In 1983 Friedman and Olson noted that the rrnB opérons of E. coli contain a region homologous to the nut site of phage X. This region, located within the 67 bp immediately upstream o f the P2 promoter, was shown to decrease transcription termination by about 50% (Li et a l, 1984). The «w/-like sequences, that comprise boxA
sequence elements, are conserved in all seven rrn opérons of E. coli. There are also boxA
sequences in the spacer region between 16S and 23S genes (Morgan, 1986). The boxA
sequence of the E. coli rrn opérons is a potent antiterminator on its own, but its function is strongly dependent of the presence of a consensus sequence 5’- UGCUCUUUAA(C/A)A-3’ (Berg g/ a/., 1989). Point mutations in the boxA reduced
1993 Nodwell and Greenblatt showed that a, heteroduplex formed by NusB and ribosomal protein SIO, interacts specifically with boxA RNA, and mutations in boxA
that impair its antitermination activity compromise its interaction with NusB and SIO (Nodwell and Greenblatt, 1993). The same authors have proposed a model in which transcription complexes synthesizing rRNA have an associated ribonucleoprotein complex containing SIO, NusB and the boxA component of the nascent transcript, held in place by the interaction of SIO with the surface o f the RNA polymerase.
Sequences with homology to the E. coli boxA motif were first identified within 65 bp downstream from the start of transcription of the rrn operon o f M.tuberculosis
(Kempsell et a l, 1992). Later, it was shown that a stretch of 31 nucleotides present in the leader region that includes the boxA signal and the RNase III cleavage sites, was identical in 8 species of slow growing mycobacteria (Ji et a i, 1994a). The intergenic spacer region between 16S and 23 S genes also contains a highly conserved region of 46 bp in which a boxA signal can be identified (Ji et al., 1994b). Possible antitermination sequences were also found to be present in rrnA and rrnB opérons of fast growing mycobacteria (Ji et al., 1994c). The mycobacterial boxA element 5’- UG(GAJ)UGUUUGA(G/U)(U/A)-3’ (Kempsell et al., 1992), is related to, but different from the boxA sequences reported in other species (see figure 5.1.). Although the mycobacterial boxA sequence is unlikely to bind the E. coli proteins NusB and SIO, it is likely to interact with the homologous mycobacterial factors.
In summary, the boxA RNA sequence plays a determinant role in transcription antitermination in rrn opérons o f E. coli by interacting with NusB and ribosomal protein
I
SIO to form a complex resistant to transcription termination. The presence o f highly conserved sequences resembling E. coli boxA in the leader and spacer regions o f the
M.tuberculosis rrn operon, suggests that transcription antitermination also occurs in mycobacteria. We were interested in defining the role of boxA signals in transcription antitermination in the rrn opérons of M.tuberculosis, particularly its interaction with the mycobacterial proteins homologous to ribosomal protein SIO and NusB. To do so it was necessary to design an experimental system to synthesize RNA molecules containing the mycobacterial boxA sequence.
Gr am- pos i t i ve : (a) M y c o b a c t e r i a ( b) S t r e p t o m y c e s (c) B. s u b t i l i s ( d) M c a p r i c o i u m 5 ’- T G ( G / T ) T G T T T 5 ’- C G T T ( C / G ) ( C / T ) T T G A ( G / T ) ( T / A ) - 3 ’ G A G A - 3 ’ 5 ’- T G ( A / T ) T C T 5 ’- ( A/ G) G A T C T T T G A A A - 3 ’ T T ( C/ G) A A A - 3 ’
Gram- negat i ve:
(e) co l i 5 ’- T G C T C T T A A( C / A ) A - 3 ’
( f ) Phage X: 5 ’- ( C / T ) G C T C T T - 3 ’
Figure 5 . 1 Comparison o f DNA sequences encoding boxA signals in rrn opérons o f
different species. The letters shown in green indicate the bases that are identical in all
the hoxA sequences. References: (a) Ji el al, 1994a, b and c; (b) Perdonet el al., 1989.
o f templates containing the DNA sequences encoding mycobacterial boxA, under the control of a bacteriophage promoter. The templates were transcribed in vitro to obtain RNA molecules carrying the pertinent antitermination signal. The in vitro transcription system is an easy and reliable technique, that can be adapted to synthesize different types o f RNA.