Functional inactivation rates of the messenger RNA molecules coding for the individual ribosomal proteins in Escherichia coli.

Summary The rates of functional decay of messenger RNA coding for total soluble, total ribosomal and individual ribosomal proteins were measured in Escherichia coli strain AS-19, at 30^o. This was accomplished by blocking RNA synthesis with the inhibitor thiolutin and measuring residual protein synthesis at various times thereafter. The data obtained expressed as a decay constant (Hartwell and Magasanik, 1963) show that both total soluble and total ribosomal protein decay with similar rates (K ₂=0.64 and 0.61 respectively) which are slightly faster than the decay rate of -galactosidse (k ₂=0.43) under these conditions. All the individual ribosomal proteins appear to comprise a population of cistrons whose individual mRNA's decay with very similar rates with the possible exception of protein L3, whose mRNA appears consistently to decay very rapidly.Additional data on the stability of the total soluble and total ribosomal proteins during thiolutin treatment (that is, proteins synthesized in the absence of concommitant ribosomal RNA synthesis) fail to demonstrate any marked difference between these two protein populations. Examination of the stability of the individual ribosomal proteins however, reveals that some are degraded up to 35% in 15 min of thiolutin exposure, some to about 15% and some appear to be completely stable. In general, a degree of correlation exists between the stability of a given protein and the observed decay rate of its messenger RNA. This observation may explain in part the spread among the rates of mRNA decay. Nevertheless, we conclude that although degradation is occurring, it is not sufficient to alter the main conclusion that the rates of functional decay of mRNA cistrons coding for the ribosomal proteins are very similar.     

The lognormal distribution fits the decay profile of eukaryotic mRNA.

A general program was written which simulates radioactive labeling of RNA $\text{in vivo}$. The program was used to determine the effect that different distributions of half-lives would have on the composite decay curve observed in a pulse-chase experiment. Four biologically relevant points emerge: 1) The published, experimentally determined composite decay curves for eukaryotic mRNA are not compatible with a normal, uniform, or exponential distribution of decay times. 2) The experimental curves are compatible with a lognormal distribution of decay times as well as the two-component discrete distribution previously hypothesized. 3) If the lognormal or some similar distribution were correct, about half the mRNA species would decay faster than what is presently called the “fast component of decay”. This point is crucial to any argument about the fraction of poly (A) or other nuclear sequence that is transported to the cytoplasm. 4) If a $\text{particular}$ mRNA species is found to decay at a constant rate for 3 half-lives, that is not only consistent with 1 half-life for all the mRNA, but also consistent with 20 different half-lives which are normally or uniformly distributed.In addition to the decay of mRNA, the lognormal distribution is also compatible with data on the decay of poly(A)-containing nuclear RNA and total cellular protein.     

Evidence for endonucleolytic cleavage at the 5''-proximal segment of the trp messenger RNA in Escherichia coli.

Summary The 5-proximal trp leader RNA segment (about 5S) decays at 2 to 3 times slower rates than the distal trp mRNA sequence. This has been demonstrated by employing the deletion mutants which lack a large portion of the structural genes but retain the promoter-proximal region of the trp operon. Relative stability of the leader RNA is not merely due to the presence of an untranslatable region in the segment; the internal untranslatable segment of trp mRNA downstream from the nonsense alteration site of a double mutant trpAD28·trpE9758 decays as fast as the normal trp mRNA sequence. These results suggest that the trp mRNA is endonucleolytically cleaved to yield the small 5-proximal leader RNA segment before the distal mRNA decays and that the leader RNA sequence is not subject to usual mode of mRNA decay in the 5 to 3 direction.     

Antisense RNA based down-regulation of RNaseE in E.coli

Background  

Messenger RNA decay is an important mechanism for controlling gene expression in all organisms. The rate of the mRNA degradation directly affects the steady state concentration of mRNAs and therefore influences the protein synthesis. RNaseE has a key importance for the general mRNA decay in E.coli. While RNaseE initiates the degradation of most mRNAs in E.coli, it is likely that the enzyme is also responsible for the degradation of recombinant RNAs. As RNaseE is essential for cell viability and knockout mutants cannot be cultured, we investigated the possibility for a down-regulation of the intracellular level of RNaseE by antisense RNAs. During this study, an antisense RNA based approach could be established which revealed a strong reduction of the intracellular level of RNaseE in E.coli.