Difference Between Functional and Structural Integrity of Messenger RNA

Abstract

Messenger RNA molecules that are structurally stable, as measured by their ability to hybridize to DNA, may nevertheless be considerably less stable in retaining their ability to function in protein synthesis. The structure of the majority of the mRNA of phage S13 decays with a half-life of 10.6 4-0.5 min. In contrast, much of the function of the mRNA that is involved in synthesis of a capsid protein (product of the F gene) decays rapidly with a half-life of 1.4 4 0.8 min; a residual amount of function decays with a half-life of 14.0 4 4.0 min. The measurements were made in the presence of rifampicin, which was used to prevent the formation of new mRNA. A proposed model for the functional decay is based on the polycistronic nature of the mRNA. Degradation of the mRNA would proceed in two steps: the first step would be a fast attack at a region near the 5'-terminus of each molecule that would eliminate the function of the proximal message; the second step would be a slow attack on the remaining messenger molecule precipitating a subsequent rapid degradation of the physical structure.Studies on degradation of messenger RNA of bacteriophages OX174 (1, 2), M13 (3), and T7 (4, 5) have shown that their mRNA is relatively resistant to decay. At least in T7 infection only the viral mRNA is stabilized, not the mRNA of the infected host, thus ruling out a viral-directed general interference with mRNA degradation.For the above three viruses, only decay of the ability of the mRNA to hybridize to DNA was measured, and no information was obtained on decay of function of the mRNA. But the functional half-life is critical in protein synthesis. Alteration or removal of perhaps just one nucleotide could rapidly eliminate the function of mRNA without significantly reducing the amount of mRNA detected by hybridization. As a result, a structurally long-lived messenger could have a quite short functional life.Since it is likely that degradation of mRNA plays an important role in regulation of protein synthesis, we have studied the decay of both biological function and structural integrity in the S13-OX174 phage system. Functional decay was studied by examination of the rate of protein synthesis during inhibition of new RNA synthesis by rifampicin. The rate of protein synthesis was measured by the uptake of a labeled amino acid during short pulses into the major phage (1, 2) and with the small filamentous phage M13 (3). We examined early and late S13 mRNA to see if both have the same decay rate, particularly to examine the possibility that after infection some phage-specified protein may be synthesized that selectively protects phage mRNA against degradation; if this were true, the early messenger that would have to code for such a protein could not be protected, and would be degraded as fast as the host mRNA. We also examined the effect of messenger concentration on its degradation by using a mutant in gene A, blocked in DNA replication, that produces four-times less mRNA than the wild type (6). Thus, we could test the possi...

Processing Endoribonucleases and mRNA Degradation in Bacteria

“…However, if one reviews the literature published when investigators were still grappling to understand the basic mechanics of mRNA degradation, it can be seen that there are a variety of conditions in which mass decay can be slower than is the rate of functional decay (51,62,109,110,117,120,141).…”

Transcriptional regulation of the yeast cytochrome c gene.

“…The results of these experiments are presented in Fig. 3, a plot of (11,12). This apparent contradiction would be resolved if the degradation of mRNA were to proceed by an initial rapid endonucleolytic cleavage of the RNA, followed by degradation of the remaining fragments.…”

Early gene expression in bacteriophage T7. I. In vivo synthesis, inactivation, and translational utilization of early mRNA's

“…Relative molar amounts of T7 early mRNA present after rifampin addition to chloramphenicoltreated cells infected at 30 C. Experimental procedures were as described in the legends to Fig. 1 and 2 except that the infection was carried out in chloramphenicol (200 Ag/ml added at 5 min before infection) at 30 C. Infected cells were labeled with [3H]uridine (34 AtCi/ml, 22 Ci/mmol) added immediately after infection, and rifampin (100 Ag/ml) was added 7 min later. Relative molar amounts of T7 early mRNA's are displayed in Table 2.…”