3.7. FACTORES DETERMINANTES EN LA EVALUACIÓN TÉCNICA
3.7.1.4. Aspectos hidráulicos de las fuentes subterráneas
One of the aims of this part of the project was to identify protein factor(s), other than ribosomai protein SIO, involved in transcription regulation in the rrn operon. As part of our efforts to do so, we attempted to isolate a NusB homologue from a pool of cytoplasmic mycobacterial proteins. The experimental set up that we have used is based on the findings of Nodwell and Greenblatt (1993) that in E. coli ribosomai protein SIO forms an heterodimer with NusB, and that this complex specifically binds to RNA containing the boxA sequence. If a similar interaction occurs in the rrn opérons o f M. tuberculosis, it would be possible to use the purified ribosomai protein SIO and boxA
interact with these two elements (details o f the methods used to identify and produce
boxA sequences are described in chapter 5).
A schematic description of the experimental design is shown in figure 4.1. Recombinant ribosomai protein SIO is incubated with a mycobacterial cell free extract, in the presence of RNA containing the boxA sequence. The mixture is then incubated with a Ni-NTA resin: the recombinant ribosomai protein SIO involved in the complex formation would be trapped by the resin, and so would be the RNA and the putative NusB protein of M. leprae. After washing the column carefully, the “complexes” are eluted from the resin using Imidazole, and the composition of the eluate is examined. Any proteins eluted together with ribosomai protein SIO are assumed to be part of the complex. The success of such experimental approach depends on a series of conditions that need to be verified before applying it as a tool to isolate transcription elongation factors: (1) there must be an interaction between the three factors (S10-NusB-6o%v4), with a relatively high constant affinity; (2) the interaction must not be affected by immobilizing the ribosomai protein SIO in the resin; (3) ribosomai protein SIO should not bind boxA RNA by itself; (4) boxA RNA should not bind to the Ni-NTA resin; (5) proteins from the cell free extract should not be captured by the Ni-NTA resin; (6) the interaction must be specific for boxA RNA. In order to answer some of these questions it is necessary to have a way of monitoring the binding of the elements. We have done preliminary experiments using a double labelling combination: on one hand, ribosomai protein SlO was in vitro transcribed/translated in the presence o f [^^S]-labelled methionine; on the other hand, the RNA containing the boxA sequence was labelled with
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[ P]. By measuring the cpm of the fractions in a scintillation counter, it is possible to follow the elution of both the ribosomai protein SIO and the RNA from the Ni-NTA column.
Figure 4.2. shows the results obtained with the double labelling system: the
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amount of [ S] and [ P] derived cpm was measured in each one o f the fractions. Panel A shows the results of incubating the ribosomai protein SIO and èox^-RNA in the absence of mycobacterial cell free extract: although there is protein eluted from the column, as judged by the increase in [^^S] counts in the elution fractions, there is not a
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^ 0 Mycohaclerial cell free ex tract
BoxA RNA 3— Incubate at 4”C for 30 minutex. I l l l l l l l l i l l +NÎ-NTA Ni--iiiiiinmi— — —
i
i
Washings +250 mM Imidazole I l l l l l l l l i l l —i
S D S - P A G E NusB-like SIOFigure 4.1.- Schematic representation o f the experimental plan designed to isolate
mycobacterial protein factors that interact with ribosomai protein SIO and hoxA RNA.
A mycobacterial cell free extract is incubated with the hoxA RNA and the recombinant
ribosomai protein SIO and the complexes purified by means o f the 6xHis tag o f the SIO.
(A) Klution pattern of‘35S SIO from N i-N T A colum ns: No cell free extract added 40000 30000 S 20000 10000 K lu tio n p a t f c m o f 3 2 P boxA R N A K ra c H o m u m b e r 8 9 10 F r a c t i o n n u m b e r 11
(B) Klution pattern o f 35S SIO from the N i-N T A colum ns: A dded cell free extract. 400tNl 30000 S S' 2(NNN) 10000 K lu tio n p a t t e r n o f J 2 P b oxA R N A -IT F r a c ti o m u m lt c r - f 8 9 10 F r a c t i o n n u m b e r 11
Figure 4.2. Elution patterns for ribosomai protein SIO ([^^S]) and ([^^P])
from Ni-NTA columns: Effect o f incubation with mycobacterial cell free extract. The
columns represent the amount o f radioactivity (cpm) present in the fractions eluted
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from the column. The main graph shows the [ S] counts, and the small plot the [ P]
counts. boxA RNA and ribosomai protein SIO were incubated together, in the absence
(A) or presence (B) o f cell free extract, and the complexes separated using the Ni-NTA
labelled RNA containing the boxA sequence does not interact by itself with either the Ni-NTA resin or the ribosomai protein SIO. A different result is obtained when cell free extract from M smegmatis is added to the incubation reaction, as shown in panel B: although the elution pattern of ribosomai protein SIO is not altered as indicated by the
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[ S] profile, there is apeak of [ P] counts that co-elute with the [ S]. This suggests that a component of the mycobacterial cell free extract is necessary for the RNA to bind to the ribosomai protein SIO in concordance with the findings of Nodwell and Greenblatt (1993).
In order to corroborate this result, a second experiment was designed, in which
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[ S] labelled ribosomai protein SIO was incubated, in the presence of M smegmatis cell
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free extract, with [ P] labelled RNA carrying either the boxA sequence or a complementary sequence (cboxA) as a negative control. If the interaction between ribosomai protein SlO-NusB and boxA is similar to the one described for E. c o li, it must be dependent on the presence of a boxA signal in the RNA. Figure 4.3. shows the results
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of such experiment. Panel A and B show the elution pattern for [ S] and [ P], obtained when M. smegmatis cell free extract was incubated with ribosomai protein SIO in the presence of RNAs containing different signals. The graph shows that the pattern
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o f elution of [ P] is not significantly altered by using an RNA molecule containing a complementary boxA sequence, suggesting that the complex formation is independent of RNA sequence: RNA molecules seem to be trapped in complexes with NusB and ribosomai protein SIO, even if they do not carry the mycobacterial boxA sequence.