Radiation target analysis has proven a useful technique for mass analysis of proteins. Although it was assumed that it would be similarly useful for analysis of RNA mass, these data indicate that it may also provide an independent method for determining local RNA structure-function relationships. The equations used here for calculation of the RNA target sizes are those previously developed for analysis of proteins (1). A general theoretical derivation, which is independent of the nature of the target molecule, has confirmed this equation (10). There are only two factors in these equations that possibly could differ for RNA: the temperature factor and the average energy deposited in each primary ionization. The same tem perature dependence of radiation sensitivity is found in a variety of synthetic polymers as in protein (11). Thus, it is probable that the sensitivity of RNA would be similar to other organic polymers. The mean energy deposition from an electron impact with a given material depends on the elemental composition of the irradiated material. The similarity of protein and RNA in this respect leads to similar calculations of theoretical energy depositions (23). Thus, these equations are expected also to be valid for RNA.
Effects of buffer on radiation target analysis of proteins has been reported (9). This is of special concern in this study. Buffered solutions containing Tris in the concentration and pH range employed in this study have been shown to stabilize nucleic acids from autocatalysis (12), in addition to being identical to those used successfully for polypeptide analysis.
Target Analysis of Structure
The smaller transcribed ribozyme is known to be approximately 78 kDa based on migration on denaturing polyacrylamide gels employing known size standards, and predicted from the restriction-cleaved template. The RNA target size of 80 kDa based on densitometric assay of the surviving 262 nt species is close to the known mass of 78 kDa. Radiation inactivation of the 1226-nt ribozyme gave a target size based on the loss of structure of 319 kDa. It is apparent that a primary ionization occurring anywhere in the polynucleotide chain results in a scission of the polymer backbone. In this respect the response of RNA is similar to that found in polypeptides (13). Previous studies of irradiated protein monomers resolved by denaturing SDS/PAGE showed that a primary ionization caused breakage of the polymer backbone so that the fragments no longer moved as intact monomers. The fragments have greater electrophoretic mobility than the native polypeptide.
Target analysis of the amount of surviving monomers yielded target sizes equivalent to the entire polymers; this indicated that a single radiation hit anywhere in a polypeptide resulted in chain cleavage. Here we find that like proteins, a radiation hit anywhere in a polyribonucleotide leads to scission of the polymer chain. Unlike polypeptides, however, the damage due to electron impact is not globally distributed throughout the phosphoribose backbone.
Target Analysis of Activity
Ribozyme molecules containing an inactive 'leader' component retain full catalytic activity (ref. 14; S.L.B., unpublished data). The 262-nt ribozyme target size estimated from activity (14.8 kDa) is close to the minimal length of the embedded ribozyme moiety (15.5 kDa) known to possess full cleavage activity (S.L.B., unpublished observations). Similarly, the 1226-nt ribozyme target size based on enzymatic activity was 15.9 kDa. Thus, increasing the total RNA strand length more than 4-fold did not significantly alter this target size. The loss of enzymatic activity reveals that the energy deposited by a primary ionization is not transmitted along the total length of the polynucleotide chain, which would result in inactivation of function, but rather is restricted to a local region. This is the opposite of observations made in proteins.
The loss of function in irradiated proteins reveals that energy deposited in a primary ionization is transferred throughout the polypeptide backbone, causing multiple ran dom damages with inactivation of the entire molecule. This complete inactivation is shown in a single protein that contains several different and spatially separated enzymatic activities; one radiation interaction destroys them all (15). It is apparent from this study that the same principal does not hold for RNA. The estimated ribozyme target size determined from activity is close to the minimal ribozyme moiety, which possesses full cleavage activity (15.5 kDa; Table 1), not to the overall mass of the molecule. Because the enzymatic activity of the RNA molecule is destroyed only when there is a radiation interaction in the local region, there can have been no radiation energy transfer throughout the polyribonucleotide that is capable of irreversible molecular damage. The close correspondence between the target masses and the known ribozyme moiety indicates that there is no detectable transfer of energy.
Kinetic analysis of the surviving ribozyme activity of ribozymes after irradiation reveals a decrease in vm,a with no detectable change in Km for the small ribozyme. As in the case of proteins, there is a decrease in the number of active molecules, but the intrinsic activity of each surviving molecule is unaltered. Therefore, as expected, RNA molecules damaged in the core ribozyme moiety have no activity.
Radiation Effects in Oligosaccharides and Glycoproteins
Previous radiation studies of glycoproteins and of oligosaccharides had shown a lack of energy transfer along the oligosaccharide chain. Irradiation of oligosaccharides results in gross damage at a single lactone ring and in the adjacent ring on either side. There is no observable energy transfer beyond that region (16, 17). Similarly, in glycoproteins, there is no transfer of radiation energy between the oligosaccharide and the attached polypeptide (18).
This study suggests that ribose in the polynucleotide chain behaves similarly to oligosaccharide sugars. The mechanism by which these ring sugars inhibit radiation energy transfer along a polymer is unknown. However, the resemblance of these structures to ascorbic acid, a well-documented free radical scavenger, may suggest a similar role for these sugars. Implications for Other Nucleic Acid Radiation Studies Previous studies on RNA target sizes based on viral infectivity yielded mass estimates comparable to the entire mass of the viral RNA. This suggests that essentially the whole nucleic acid molecule is required for viral replication and infectivity. In this study, radiation target analysis was found useful in determining the mass associated with a local RNA function. It may be valuable for studying other RNA functions and interactions, such as defining the oligoribonucleotide length in ribonucleoprotein structure and RNA inter- and intrastrand associations resulting in secondary and tertiary nucleic acid structure. If the radiation resistance of RNA is due to the lactone ring as shown to be the case with oligosaccharides (16), a similar response would be predicted for DNA. Thus, radiation may be useful in transcriptional start-site analysis and understanding the role played by specific oligonucleotides in determining basal and extended transcriptional complexes (19). Finally, it is obvious that RNA radiation sensitivity is much less than that for polypeptides. The earliest life forms have been postulated to have been based on RNA rather than protein (20-22). Thus, if the primordial world had a high radiation exposure level, life forms based on nucleic acid would have had a selective advantage in activity and survival over polypeptide-based forms of life.
Answers to all your Biology Questions
