Abstract
The stability of purified poliovirus RNA in cell-free translation systems prepared from HeLa cells or rabbit reticulocytes has been examined. Degradation of the RNA occurs with a t1/2 of approximately 35 min at 30 degrees C under conditions used for in vitro translation. Degradation is due in part to activity in the cell lysate, and in part to contaminants in the commercial preparations of creatine phosphokinase used in the energy-regenerating system. Addition of crude preparations of initiation factors significantly slows degradation, presumably as a result of protein-RNA interactions which confer resistance to nuclease action. Prior treatment of RNA with methylmercury hydroxide has no effect on degradation rates. On the other hand, endogenous mRNA, present as a messenger ribonucleoprotein particle in extracts from poliovirus-infected HeLa cells, remains completely intact during in vitro translation. These infected cell extracts synthesize the normal complement of viral proteins and utilize two different initiation sites for translation. Treatment of the infected cell extract with micrococcal nuclease destroys the endogenous mRNA. Subsequent addition of exogenous RNA to the same extract results in the formation of a protein-associated RNA particle with sedimentation properties slightly different from the endogenous messenger ribonucleoprotein, and the added RNA is unstable. We conclude that two initiation sites can be utilized on intact poliovirus mRNA, and fragmentation of the RNA is not prerequisite for generation of a second site in this RNA.
Highlights
Degradation of Polio RNA in Protein-synthesizing Systems-Translation of exogenous mRNAs in a variety of extracts prepared from rabbit reticulocytes, ascites tumor cells, or cultured mammalian cells has become a standardprocedure for numerous types of studies in many laboratories
We have previously used such extracts, prepared from cultured HeLa cellsor from rabbit reticulocytes, to study translation of poliovirus RNAin vitro, and we have shown accurate and efficient production of poliovirus proteins (21; see Fig. 3, below)
Associations between mRNA and proteins in initiation factor preparations have been reported previously, and we have demonstrated an increase in the sedimentation coefficient of other mRNAs after incubation with initiation factors in a translation reaction [20]
Summary
From the Departments of Cellular, Viral and Molecular Biology and Biochemistry, University of Utah Medical Center, Salt Lake City, Utah 84132. Incubation of mRNA in cell-free extracts has been reported to result in degradation These infected celelxtracts synthesize the normal com- [18].RNA fragmentation might conceivably precede plement of viral proteins and utilize two different ini- and be required for initiation at multiple sites. Several considerations have led to proposals such asthe "scanning model" presented by Kozak [1], in which a 40 S ribosomal subunit (with associated initiation factors and initiator tRNA) binds initially at or near the 5' terminus of a mRNA and subsequently migrates until the first AUG codon is encountered This type of mechanism is consistent with the observed monocistronic character of most eukaryotic mRNAs, and with the facilitating effect of the 5'terminal m7G cap group, regardless of the distance between that cap and theinitiator codon
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
