Agauugcaa G A

Vu UCOGCCC"

UBCDfllJICt1

Figure 9. Summary of sites protected in RNase P RNA from E. coli (A) or B. subtilis (B) by the protein subunit. Sites protected against either RNAse Tl98 or DMS91 in the E. coli RNA or against Fe(II)-EDTA in the B. subtilis RNA56 are shown in black.

to several non-contiguous regions of the RNA (Figure 9), which is different from the mode of binding of several other RNA-binding proteins. It is possible that these potential binding regions are close enough in space to constitute a single binding site or are independently bound by different RNA-binding sites in the protein. We described above three possible binding sites in the crystal structure of the RNase P protein: the protection data, however, are difficult to interpret unambiguously in terms of protein-RNA contacts. Other interpretations are possible, such as conformational changes in the RNA upon binding to the protein. A three-dimensional model of the interaction between the protein and the RNA subunits of bacterial RNase P has yet to be developed.

9.4.2 The Protein Subunit as a Cofactor

In analyzing what the function of the protein subunit in catalysis could be, one must consider first what the kinetic parameters of the reaction affected by the protein are. The reaction catalyzed by the holoenzyme is more efficient (Table 2) than that of the RNA alone, due mainly to effects on kcM. The protein subunit of RNase P alleviates the need for high ionic strength of the RNA-alone reaction. The rate-limiting step in the RNA-alone reaction is product release.78'93 This is revealed by the fact that under single turnover conditions (excess enzyme) the first round of substrate cleavage is much more rapid than subsequent rounds. In the holoenzyme, there is no difference in the rates of the first and subsequent rounds of cleavage. Also, mature tRNA behaves as an inhibitor of the RNA-alone reaction, but in the presence of the protein inhibition is much reduced. These data resulted in the suggestion78 that the highly positive protein subunit acts as a localized electrostatic shield facilitating the interaction between the catalytic RNA and the substrate without interfering with rapid product release. In the RNA-alone reaction the high ionic strength required for efficient catalysis provides the shielding effect but this is not localized and product release is slowed.

The function of the protein subunit in bacterial RNase P might be more complex and subtle than just that of a localized electrostatic shield. When the function of the protein is analyzed in a more detailed way, additional interesting features appear. For instance, for some substrates, the protein reduces the Km several fold.41 In vivo experiments suggest that the protein has a differential effect on the processing of different substrates.42 Recent detailed studies in the B. subtilis system suggest an important role of the protein subunit in catalysis through interaction with the 5' leader of pre-tRNAs.16'51'56'66 While yet to be definitively established, these interactions may be mediated by the central cleft in the protein (Figure 8).

The protein subunit of RNase P increases the versatility of the RNA subunit. Mutants in the RNA subunit that have reduced catalytic activity in the absence of protein have wild-type-like behaviour in the presence of the protein.57 Using in vitro selection techniques this idea has been subjected to a direct test.40 The sequence of the bottom part of the highly conserved helix P4 of E. coli RNase P RNA was randomized and active ribozymes selected in the presence or absence of the protein cofactor. In the absence of protein only the wild-type sequence of the RNA was recovered, while in the presence of protein several additional sequences were selected. These mutant RNAs were active only in the presence of the protein. The protein subunit also increases the range of substrates that can be processed. While the RNA alone can efficiently process only pre-tRNAs, the holoenzyme can process a number of additional substrates. This question will be discussed in the next section in the context of evolution of RNase P.

9.4.3 Protein Subunits in Eukaryotes and Archaea

Aside from their identification and sequencing, very little is known of the function of the protein subunits in eukaryotic RNase P.10'39'87'88 To date no eukaryotic RNase P holoenzyme has been reconstituted from purified RNA and protein subunits. The development of such an in vitro system would be an important step forward in the characterization of eukaryotic RNase P.

RNase P from Archaea is intriguing. Several complete archaeal genome sequences are available and a protein homologue to a nuclear RNase P protein has been identified.32 Chimerical holoenzymes have been reconstituted from some archaeal RNase P RNA and bacterial RNase P protein65'70 and from E. coli RNA and archaeal protein, in one case.54

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