Kan Agarwal

Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS.

TRANSCRIPTIONAL elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex1—4. In prokaryotes its regulation pro-vides an important mechanism of genetic control1. Analogous eukaryotic mechanisms are not well understood5, but may control expression of proto-oncogenes6,7 and viruses, including the human immunodeficiency virus HIV-1 (ref. 8). The highly conserved euk-aryotic transcriptional elongation factor TFIIS9 enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins10—14. Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through10 and possibly nascent transcript cleavage11—15. Here we describe the three-dimensional NMR16 structure of a Cys4 nucleic-acid-binding domain from human TFIIS9—10. Unlike previously characterized zinc modules17—21, which contain an a-helix, this structure consists of a three-stranded β-sheet. Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA trans-actions22—24, including RNAPII itself25. This new structure, desig-nated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures26 and highlights the growing recognition of the β-sheet as a motif of nucleic-acid recognition27—28.

Catalysis of DNA joining by bacteriophage T4 RNA ligase

Abstract RNA ligase, purified extensively from Escherichia coli infected with wild-type or DNA ligase mutants of bacteriophage T4, catalyzes the joining of 5′-phosphoryl terminated DNA to DNA and RNA acceptors. This was shown by the conversion of [5′-32P]deoxyoligomers to a form resistant to phosphatase, the increased chain length of the joined RNA-DNA copolymers, the circularity of the DNA joined product, and the transfer of the 5′-32P label of the donor DNA to the 3′-end of both RNA and DNA acceptors. The novel DNA joining activity is intrinsic to RNA ligase since it co-purifies with RNA joining activity and has the same requirements, inhibitors, and thermolability. These results suggest that RNA ligase should be a useful reagent for the synthesis of defined sequence DNA and RNA-DNA copolymers and raise the possibility of a role for RNA ligase in DNA metabolism.

N,N-Bis[2-oxo-3-oxazolidinyl]phosphorodiamidic chloride: a novel coupling reagent in the synthesis of oligodeoxyribonucleotides

Abstract Use of a novel coupling reagent, N,N-bis[2-oxo-3-oxazolidinyl]phosphorodiamidic chloride (BOPDC) in the synthesis of oligodeoxyribonucleotides is described. This reagent allows the synthesis of phosphotriesters in high yields (70–80%) without detectable side reactions. Synthesis of a hexanucleotide, d(A-A-C-C-C-G) is presented as an example.

Interaction of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with relaxed circular pBR322 DNA.

The interaction of (+/-)-BPDE (1) with DNA at neutral pH was investigated by the application of relaxed circular pBR322 DNA. (+/-)-BPDE causes a rapid positive supercoiling of this DNA followed by a slower spontaneous relaxation. The results indicate that there are two clearly discernible types of chemical interactions between 1 and DNA, a rapid intercalative covalent binding and a slower strand breakage. The implications of these findings are discussed.

Rat gastrin's amino acid sequence determined from the nucleotide sequence of the mRNA ☆

Abstract Gastrin cDNA was synthesized using a synthetic oligodeoxynucleotide to specifically prime rat mRNA preparations for reverse transcription. Hybridization analysis using this cDNA indicated that rat gastrin mRNA is the same size as that which codes for hog gastrin. The amino acid sequence for rat gastrin 34 was determined from a partial nucleotide sequence analysis of the cDNA and indicates that in rat, as in other species, gastrin is synthesized as a larger precursor which is processed following two consecutive basic amino acids to yield molecules of 34 and 17 amino acids (gastrin-34 and gastrin-17).

Separation of diastereomers of methylphosphonate dinucleotides

Abstract A chiral derivatizing agent, 1-menthyl chloroformate, has been used as a 3′-OH blocking group to facilitate the resolution of diastereomers of methylphosphonate dinucleotides by silica gel column chromatography.

STUDIES ON GASTRIN mRNA STRUCTURE USING AN OLIGONUCLEOTIDE PROBE

A detailed understanding of the regulatory mechanisms of gene expression in higher organisms depends upon the detection and isolation of genes and genespecific mRNA. However, progress in the isolation of many mRNAs has been hampered by their low levels within the cellular RNA pool. In addition, the size distribution of mRNA molecules in most cells is such that their sedimentation values overlap one another and are close to those of ribosomal components. Thus, a very sensitive assay procedure is needed for the isolation of a specific mRNA. Generally, assays for the detection, isolation, and characterization of cytoplasmic RNAs have involved use of cell-free translation systems in conjunction with immunoradiometric assays to identify specific protein products. This approach may not be suitable, however, for study of mRNA species directing the synthesis of proteins that undergo extensive post-translational modification. In such cases, the available antibodies are usually directed against the processed protein and may not interact efficiently with a primary translation product synthesized in vitro. Furthermore, the amount of a particular mRNA present in the total cellular mRNA pool may be so low that detection using established procedures may not be possible. t o overcome these problems, which we encountered in our studies with gastrin, we have developed a sensitive alternative approach for studying mRNAs coding for proteins which may be rapidly processed in vivo. This approach is highly sensitive and allows the detection of as little as 0.2 fmol of gastrin mRNA in a preparation of poly(A)-RNA from hog antrum.’ This method should be of general application for detection and characterization of mRNAs corresponding to proteins of known amino acid sequence. The principle of the approach is simple and involves the use of a deoxyoligonucleotide probe specific for a particular mRNA. The nucleotide sequence of the probe can be deduced from the known amino acid sequence of the corresponding protein. Because of the degeneracy in the code, the amino acid sequence selected should be such that the deduced nucleotide sequence of the probe contains a minimum number of nucleotide mismatches. For studying gastrin mRNA, which has served as a model system for the development of this approach, the unique amino acid sequence TrpMet-Glu-Glu (see FIGURE 3), was selected for the deduction of the nucleotide sequence of the DNA probe. The sequence of the dodecanucleotide d(C-T-C-C-T-CC-A-T-C-C-A) deduced from the tetrapeptide could contain a maximum of two mismatched base pairs because there are two possible codons for glutamic acid, GAA and GAG. We selected the codon GAG in designing the gastrin primer because G in the third position is statistically favored for all codons,‘ and, in particular cases in which known amino acid sequences have been correlated with determined mRNA sequences, Glu is usually coded for by the trinucleotide GAG.’ If one or both of the