RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein.We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPRI (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPRI 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPRI RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPRI transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPRI-disrupted haploids with one variant of RPRI gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPRI RNA is an essential component of this enzyme.RNase P from both procaryotic and eucaryotic sources is an endonuclease that cleaves pre-tRNA substrates to yield mature 5' termini. In procaryotes, both the RNA and protein components of the ribonucleoprotein enzymes are required for in vivo activity (15,27,29,33,43,49,57,58), but the RNA component alone is capable of efficiently catalyzing the correct reaction under some conditions in vitro (3,18,21,22,52). Extensive phylogenetic sequence comparisons of these RNAs (30), combined with folding energy calculations and cleavage sensitivity studies (6, 19, 51, 52), suggest a conserved, highly ordered secondary structure. Most if not all of the key contacts with the substrates depend on this structure, with the protein contributing to efficiency through secondary effects such as charge shielding between RNA chain phosphate backbones. The mechanism by which pretRNA substrates are recognized is not clearly understood, however, since there are no obvious regions of required Watson-Crick base pairing between the enzyme and substrate RNAs (20,36).Studies of eucaryotic RNase Ps have suggested that they also contain essential RNA subunits, although the role of the RNA components has not been firmly established. RNAs that copurify with both nuclear and organelle RNase Ps have been characterized (1,7,9,11,12,14,24,32,34,35,37,38,40,42) and shown to have sequence and structural similarities to each other and to a lesser degree to the procaryotic RNAs (5, 37). The identification of the eucaryotic RNAs as essential RNase P subunits has not been rigorously confirmed, however, since there are no reports of the eucaryotic RNAs having enzyme activity in the absence ...

Several enzymatic and chemical reagents were used to probe the secondary structure of Saccharomyces cerevisiae nuclear RNase P RNA in the presence and absence of its protein components. Double-stranded regions were detected with RNase V1 and single-stranded regions with RNase ONE (Escherichia coli RNase I). Nucleotides not paired at Watson-Crick positions were monitored with dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-[2-(N-methylmorpholinio)ethyl]carbodiimide p-toluenesulfonate. The results supported most aspects of the previously proposed, phylogenetically-derived RNA secondary structure, although minor refinements allowed incorporation of both the biochemical and phylogenetic data. Digestion of the RNase P protein(s) with proteinase K gave enhanced reactivities to structure probes at selected positions, indicating regions of the RNA made inaccessible by the presence of the protein subunit(s). The regions of RNA protected in the yeast nuclear holoenzyme were considerably more extensive than that seen in the Escherichia coli holoenzyme, consistent with the observation that the protein moiety generally comprises a larger percentage of the RNase P holoenzyme in eukaryotes than in eubacteria.

Folic acid plays a central role in anabolic metabolism by supplying single-carbon units at varied levels of oxidation for both nucleotide and amino acid biosyntheses. It has been proposed that 5-amino-4-imidazole carboxamide riboside 5'-triphosphate (ZTP), an intermediate in de novo purine biosynthesis, serves as a signal of cellular folate stress and mediates a physiologically beneficial response to folate stress in Salmonella typhimurium (B. R. Bochner, and B. N. Ames, Cell 29:929-937, 1982 In this strain, histidine represses the synthesis of both ZMP and ZTP. Treatment of cells of this strain with trimethoprim resulted in a decrease in the folate/protein ratio of cell extracts, but a blockade of Z-ribonucleotide accumulation did not affect the extent of folate depletion seen in treated cells and had only a small effect on the resistance of this strain to growth inhibition by trimethoprim. The patterns of protein expression induced by treatment of this strain with trimethoprim or psicofuranine were examined by two-dimensional electrophoretic resolution of the total cellular proteins. No differences in protein expression were seen when the treatments were performed in media containing or lacking histidine. These studies failed to provide evidence in E. coli for a folate stress regulon controlled by ZTP.

We have designed a skills matrix to be used for developing and assessing undergraduate biochemistry and molecular biology laboratory curricula. We prepared the skills matrix for the Project Kaleidoscope Summer Institute workshop in Snowbird, Utah (July 2001) to help current and developing undergraduate biochemistry and molecular biology program designers to determine which laboratory techniques, skills, and theories to include in a 4-year plan. The skills matrix can be used to evaluate and assess the types of laboratory skills as well as the level at which they are taught in biochemistry and molecular biology curricula. The matrix can foster better communication between faculty in chemistry, biology, math, and physics as they share curricular information. As an example of utility of the skills matrix, we used it to survey several commonly used biochemistry laboratory manuals to evaluate the skills covered in each text.Keywords: Matrix, assessment, undergraduate, biochemistry, laboratory, skills.Developing an appropriate undergraduate program in biochemistry and molecular biology (BMB) 1 is a challenging and ever-changing venture. Program curricula must be diligently and rigorously adjusted to reflect the rapid changes and advances in research, technology, methodology, software, and internet resources used by the discipline. New strategies and requirements for assessment make it critical to have available tested methods of evaluating programs and student learning.A solid BMB program must be built out of core courses offered by biology, chemistry, math, and physics departments. Due to the truly interdisciplinary nature of BMB programs, faculty communication, "ownership" issues, student needs, and campus limitations often dictate, or at least have an impact on, curriculum design and implementation. Luckily, there can be significant flexibility in the number and types of courses offered as long as certain guidelines are maintained. Guidelines to help direct program development and assessment are provided by organizations such as the American Association for Biochemistry and Molecular Biology (ASBMB), the American Chemical Society (ACS) and the International Union of Biochemisty and Molecular Biology [1][2][3]. Thus, the development of a BMB program course curriculum, theory, and knowledge is relatively straightforward. However, it is often a more difficult task to plan and develop laboratory strategies and mechanisms to teach students specific techniques and promote laboratory problem-solving skills. Several issues related to laboratory curricula must be considered.A plan should begin with a statement of the program's goals and objectives. It is impossible to teach all the skills and theories applicable to all of biochemistry and molecular biology in a standard one-or two-semester BMB laboratory sequence [4]; however, programs can select particular areas in which they excel, incorporate departmental strengths, and choose experiences best suited for their student population. For example, many midwestern U. S. schoo...