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Basic molecular biology

©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

I D. BASIC MOLECULAR BIOLOGY

Before getting into a discussion of genetics per se, there are a few terms of molecular biology that need to be defined in order to understand how altered genotypes cause altered phenotypes. (The following definitions, while necessary and useful for this text, are great oversimplifications. See other sections of the Microbiology Textbook for more complete definitions.) Again, a gene is the region of DNA that encodes a product. Regardless of the nature of the ultimate product (RNA or protein), this implies a promoter where RNA polymerase will bind and begin transcription and a termination signal or region where the polymerase stops (recognition of such sites may require a protein factor, such as Rho). Typically there are regulatory regions in the DNA in the vicinity of the promoter that bind regulatory proteins and that can stimulate or inhibit polymerase binding and/or transcription into the region of the gene. If the gene product is a protein, then there will also be sites signaling ribosome binding, while the end of the gene will contain signals for termination of protein synthesis with concomitant release of the completed polypeptide. Often, at least in bacteria, a unit of transcription encodes several gene products and is termed an operon.

There are several sorts of mutations that can cause a premature termination of mRNA synthesis. If this occurs in an "upstream" gene of a transcript, then it will deleteriously affect the synthesis of "downstream" gene products. Such an effect is termed polarity. This phenomenon is important in bacterial genetics because we want to identify the functions of specific gene products by examining the phenotype of mutations in those genes. For easy interpretation, we therefore want the phenotype to result from the altered gene product, not from polar effects on downstream genes.

The first class of polar mutations involves an "insertion" of a foreign piece of DNA which contains an RNA termination signal. Such a mutation eliminates downstream gene expression since no RNA polymerase will read through the "new" termination signal to reach those genes. One therefore typically sees a complete cessation of downstream mRNA synthesis. Geneexpression typically refers to the act of transcription of the gene in question.

The second class of polar mutations are those whose primary effect is the premature termination of protein synthesis. This often leads indirectly to a partial reduction of downstream mRNA synthesis. Such polarity requires the presence of a functional Rho protein, which is normally involved in transcription termination at the end of operons. A model explaining the mechanism by which premature protein stop signals, termed nonsense mutations, cause polarity is depicted in figure 2: Rho factor causes RNA polymerase to terminate transcription downstream from a site of translation termination if the following conditions exist:

1. A region of the mRNA, lacking ribosomes because of the upstream nonsense signal, contains a site that activates the ATP synthase activity of Rho, probably by binding Rho. There is in vitro evidence that such sites are C- rich and G-poor (in the mRNA) and that an approximately 12-base spacing of C residues may be important, though a precise consensus is unclear. This latter number is intriguing since biochemical characterization suggests that Rho complexes with about 13 nucleotides per protein subunit of the hexamer.

2. At some distance downstream (deliberately vague, but probably notimportant) from this site, the activated Rho "catches up" with RNA polymerase (at a polymerase pause site?) and causes it to terminate transcription.

This effect requires that there has not been translation initiation, proper or improper, between the "upstream site" and the termination region, or else ribosomes will mask the sites on the mRNA. It seems that termination sites occur fairly frequently and that the "upstream sites" may be the limiting factors in determining where polarity occurs.

The model is the following: if there is untranslated, unstructured mRNA, then there is a probability that Rho will bind there (the probability will be bigger as the "open" mRNA gets longer and when it has sequences that are preferred by Rho). Having bound, there is a probability that it will catch up with the transcribing RNAP and cause termination (the probability depends on whether or not the RNAP has passed through another ribosome binding site and loaded a ribosome, since this ribosome will prevent Rho from "sliding" up to the RNAP and terminating).

For the purpose of this text, we will make the occasionally correct assumption that the critical rho binding sites are relatively frequent and that the degree of polarity is approximately a function of the distance from the translation stop site to the translation start site of the next gene (greater distance would therefore imply more likely transcription termination.)

The effect of polarity due to this mechanism (translational stop signals) can range from very strong to negligible (0-90% reduction in downstream mRNA synthesis). Presumably the strength of the effect is a function of a set of variables: the strength of the pause site; the length and potential structure of "open" mRNA between the nonsense signal and the pause site; and the presence and strength of new ribosome binding sites following the nonsense signal which would block Rho binding.

Please do not confuse transcription with translation (or transcription terminators with translational stop signals). A transcriptional terminator will necessarily eliminate downstream translation as there will be no mRNA to translate. Many, but not all, translational stop sites will lead to a reduction of downstream transcription through the action of Rho.

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