MadSci Network: Molecular Biology |
Sharon, The answer to your question is related to the difference in the lifetime of individual DNA and RNA molecules in a cell, and how their longevity is maintained or regulated. DNA is first and foremost a medium for storing genetic information; individual molecules of DNA survive for the duration of a cell's lifecycle. Damage is repaired by the appropriate repair mechanisms, but by and large an individual DNA molecule must remain unchanged. On the other hand, the longevity of RNA in vivo is quite short, relative to DNA. While ribosomal RNA (rRNA) may be long-lived, as an integral component of the ribosomes, other RNAs often are actively degraded within minutes of being transcribed. This is especially true in regards to messenger RNA (mRNA). The discussion is complicated by the fact that control mechanisms differ between prokaryotic systems (bacterial) and eukaryotic systems (higher organisms). In bacteria, mRNA is transcribed and translated in a single cellular compartment, allowing for the two process to occur simultaneously. Before transcription is completed, ribosomes bind to the growing mRNA and begin translation. After a short period of time, RNA endonucleases begin to nick the mRNA at the 5' end, preventing additional ribosomes from binding, even before transcription or translation is completed. Generally, only a few protein molecules are made from a single mRNA before it is degraded, with the whole process lasting only a few minutes. In a eukaryotic cell transcription and translation are physically separated process, with mRNA synthesis and processing occur in the nucleus, and with translation occurring in the cytoplasm. The lifetime of mRNA in eukaryotes is typically on the order of hours to a day rather than minutes, but is still quite short compared to DNA. The comparatively short lifetime of mRNA, relative to DNA, is believed to have evolved as a means of regulating rates of protein synthesis. Remember that rates of protein synthesis are a function of both translational activation and inactivation. Translational activation is a fairly complicated subject and varies depending on the system. In general, transcription of mRNA is initiated through binding of transcription factors to promoter sequences on DNA, followed by recruitment of the appropriate RNA polymerase. The pre-mRNA product of transcription is processed (introns are excised, the 5'-end is capped, the 3'-end is polyadenylated), the mature mRNA is transported to the cytoplasm, then translated by the ribosomes. On the other hand, down-regulation of translation is universally a function of the lifetime of mRNA. In both prokaryotes and eukaryotes, translation of a particular gene is arrested through degradation of mRNA by RNases. Once in the cytoplasm, protein will continue to be translated from a single mRNA until the mRNA is actively degraded. The overall rate of protein biosynthesis is therefore a balance between RNA synthesis and degredation. It is commonly accepted that RNA degredation occurs at a relatively fixed rate, with transcriptional control being mediated by ragulation of RNA synthesis. RNases are ubiquitous in the cell, at relatively constant concentrations, giving rise to a uniform rate of mRNA degredation within a cell independent of the protein sequence a particular RNA encodes. The process and rates of RNA degradation of prokaryotic and eukaryotic systems differ in detail, but are superficially similar. RNases are of two basic types; endonucleases can cleave in the middle of an RNA molecule, while exonucleases degrade RNA from the end of a molecule. Initially, RNAs are protected from being degraded by exonucleases due to the presence of end-capping motifs. Therefore, regulated degradation of an RNA molecule begins by a cleavage event from an RNA endonuclease producing an unmodified free end, which is quickly targeted by RNA exonucleases. The precise end-capping motifs and the time-frame for RNase activity again vary between prokaryotes and eukaryotes, but the basic architecture is the same. Note that RNases exist that have other specific functions, such as RNase H, which acts to cleave the RNA component of RNA-DNA hybrids during DNA replication. Common DNases are generally those that are unique to prokaryotes, though eukaryotic DNases are known to exist (in the nucleoside, for example). DNases are generally also classified as having either exonuclease or endonuclease activity. In prokaryotes, their function is typically one of defense, aiding in the degradation of foreign DNA. Briefly, bacteria methylate their DNA as a means of differentiating their own DNA from foreign DNA. Methylation protects a DNA from being cleaved by DNases with endonuclease activity, termed restriction endonucleases or restriction enzymes. The process of methylation occurs slowly, relative to the rate of DNA replication. The methyl group serves as a tag so the cell can recognize which strand of DNA was the parent strand (the methylated one) from the daughter strand (non-methylated). In the case of a replication error, the DNA damage control proteins know to change the base on the new strand of DNA as it has not yet been methylated. Similarly, when foreign DNA invades a cell, it generally is not methylated. In the end, the restriction endonucleases serve to nick either foreign DNA or a segment of DNA containing replication errors, followed by degradation of the nicked DNA by DNA exonucleases. In eukaryotes, the DNA damage repair machinery is a bit more sophisticated, and a bit outside the scope of this discussion, so I won't go into it here. While doing some background reading to help answer your question, I did learn that two of the more common eukaryotic DNases, DNAse I and DNAse II, are thought to be involved in apoptosis. Presumably, their function is one of fragmenting chromosomal DNA beyond repair while other factors involved in apoptosis (such as ubiquitin and its function in directed protein degradation) are shutting down other major cellular functions, all of which lead to cell death. In summary, RNases are ubiquitous and are continuously degrading RNA in a cell. DNases are not as prevalent and are needed only intermitently in the life cycle of a cell. The empirical observation is, when working with RNA, occasional RNase contamination is observed any time protein contamination may have ocurred. From personal experience, it is true that RNases have appeared in my own samples, apparently through dust, cat hair, fingers, or the wind. I don't know of any occasion when similar DNase contamination may have occurred. Suggested reading: "Genes VII", Benjamin Lewin, New York: Oxford University Press, Call Number: QH430.L487 1999 "Molecular cloning : a laboratory manual", Maniatis, Fritsch, and Sambrook. Cold Spring Harbor Laboratory Press, Call Number: QH442.2 .M26 1982 "The RNA world" edited by R. F. Gesteland, T. R. Cech, J. F. Atkins. Cold Spring Harbor Laboratory Press, N.Y., c1999. Call Number: QP623 .R6 1999 Good luck with your experiments, Dr. Jim Kranz
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