Single-strand-specific nucleases, which act on single-stranded nucleic acids and single-stranded regions in double-stranded nucleic acids, are multifunctional enzymes and are ubiquitous in distribution. They find wide application as analytical tools in molecular biology research, although enzymes such as P1 nuclease are also used for production of flavor enhancers such as 5' IMP and 5' GMP. Because these enzymes are mainly used as analytical tools, very little attention was paid to aspects relating to their structure-function relationships. However, during the last few years considerable developments have taken place in this area. Single-strand-specific nucleases, their purification, characteristics, biological role, and applications have been reviewed.
The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF; EC 2.1.2.9) is important for the initiation of protein synthesis in eubacteria and in eukaryotic organelles. The determinants for formylation in the tRNA are clustered mostly in the acceptor stem. As part of studies on the molecular mechanism of recognition of the initiator tRNA by MTF, we report here on the isolation and characterization of suppressor mutations in Escherichia coli MTF, which compensate for the formylation defect of a mutant initiator tRNA, lacking a critical determinant in the acceptor stem. We show that the suppressor mutant in MTF has a glycine-41 to arginine change within a 16-amino acid insertion found in MTF from many sources. A mutant with glycine-41 changed to lysine also acts as a suppressor, whereas mutants with changes to aspartic acid, glutamine, and leucine do not. The kinetic parameters of the purified wild-type and mutant Arg-41 and Lys-41 enzymes, determined by using the wild-type and mutant tRNAs as substrates, show that the Arg-41 and Lys-41 mutant enzymes compensate specifically for the strong negative effect of the acceptor stem mutation on formylation. These and other considerations suggest that the 16-amino acid insertion in MTF plays an important role in the specific recognition of the determinants for formylation in the acceptor stem of the initiator tRNA.
Nonsense or frameshift mutations, which result in a truncated gene product, are prevalent in a variety of disease-related genes, including APC (implicated in colorectal cancer), BRCA1 and BRCA2 (breast and ovarian cancer), PKD1 (polycystic kidney disease), NF1 and NF2 (neurofibromatosis), and DMD (Duchenne muscular dystrophy). Such chain-truncating mutations can be detected using the protein truncation test (PTT). This test is based on cell-free transcription and translation of either PCR-amplified portions of the target gene or RT-PCR amplified target mRNA, followed by analysis of the product(s) for shortened polypeptide fragments. However, conventional PTT is not easily adapted to high-throughput applications because it involves SDS-PAGE followed by autoradiography or western blotting. It is also subject to human error, as it relies on visual inspection to detect the mobility of shifted bands. To overcome these limitations, we have developed a high-throughput solid-phase protein truncation test (HTS-PTT). HTS-PTT uses a combination of misaminoacylated tRNAs, which incorporate affinity tags for surface capture of the cell-free expressed protein fragments, and specially designed PCR primers, which introduce N- and C-terminal markers for measuring the relative level of shortened polypeptides produced by the chain-truncation mutation. After cell-free translation of the protein fragments, capture and detection are accomplished in a single well using a standard 96-well microtiter plate enzyme-linked immunosorbent assay (ELISA) format and chemiluminescence readout. We demonstrate the use of the technique to detect chain-truncation mutations in the APC gene using DNA or RNA from cancer cell lines as well as DNA of individuals diagnosed with familial adenomatous polyposis (FAP). HTS-PTT can also provide a high-throughput method for noninvasive colorectal cancer screening when used in conjunction with methods of enriching and amplifying low-abundance mutant DNA.
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