The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity.

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

Influence of theophylline on interferon production and cAMP level Lpa cells.

In the presence and absence of methionine initiator tRNA (tRNA(iMet)) theophylline applied for 1 h starting 6 h after induction increased significantly (2-fold) interferon (IFN) yield. Theophylline did not change the kinetics of IFN production in the presence of tRNA(iMet). The shut-off stage was present and the production was prolonged to 24 h after IFN induction. In comparison with the values obtained in untreated cells, poly I:C plus DEAE dextran (inducing complex) caused an about 2-2.5 fold increase of cyclic adenosine 3'5'-monophosphate (cAMP) level. The similar results were observed in the cells treated with inducing complex plus tRNA(iMet). About 2-fold higher cAMP level occurred in the cells under the influence of the inducing complex plus theophylline in comparison to the level obtained without theophylline. The addition of tRNA(iMet) did not influence the above presented data.

Site-directed cross-linking of mRNA analogues to 16S ribosomal RNA; a complete scan of cross-links from all positions between '+1' and '+16' on the mRNA, downstream from the decoding site.

mRNA analogues containing 4-thiouridine residues at selected sites were used to extend our analysis of photo-induced cross-links between mRNA and 16S RNA to cover the entire downstream range between positions +1 and +16 on the mRNA (position +1 is the 5'-base of the P-site codon). No tRNA-dependent cross-links were observed from positions +1, +2, +3 or +5. Position +4 on the mRNA was cross-linked in a tRNA-dependent manner to 16S RNA at a site between nucleotides ca 1402-1415 (most probably to the modified residue 1402), and this was absolutely specific for the +4 position. Similarly, the previously observed cross-link to nucleotide 1052 was absolutely specific for the +6 position. The previously observed cross-links from +7 to nucleotide 1395 and from +11 to 532 were however seen to a lesser extent with certain types of mRNA sequence from neighbouring positions (+6 to +10, and +10 to +13, respectively); no tRNA-dependent cross-links to other sites on 16S RNA were found from these positions, and no cross-linking was seen from positions +14 to +16. In each case the effect of a second cognate tRNA (at the ribosomal A-site) on the level of cross-linking was studied, and the specificity of each cross-link was confirmed by translocation experiments with elongation factor G, using appropriate mRNA analogues.

Evaluation of the use of antisense tRNA(met) as an inhibitor for eukaryotic protein synthesis.

We attempted to explore the use of antisense RNAs against tRNA as an inhibitor of eukaryotic protein synthesis. For this purpose, antisense RNA against the 5'-end half of the initiator tRNA of wheat germ was synthesized, and its effect on translation of the Brome mosaic virus mRNA was investigated in a wheat germ cell-free system. When the antisense RNA against the 5'-end half of the initiator tRNA including the anticodon sequence was added at the concentration of 8 microM to the cell-free system, protein synthesis was completely inhibited. This inhibitory effect could be suppressed by the addition of wheat germ tRNA. In contrast, sense and control RNA showed slight inhibitory effects, which were not, however, suppressed by wheat germ tRNA. The antisense tRNA formed a double-stranded RNA duplex with the target methionine tRNA in the wheat germ extract which became resistant to ribonuclease treatment. These experiments suggest that antisense tRNA could be utilized for control of tRNA functions and to block protein synthesis.

Control of initiation of eukaryotic protein synthesis by guanine nucleotides and methionyl-tRNAi.

The influence of changing concentrations of GDP, methionyl-tRNAi, eIF-2 and eIF-2B on possible rates of initiation of protein synthesis have been explored in calculations based on previously derived rate constants for interaction of the components involved in formation of ternary or quaternary complexes of eIF-2B, eIF-2, GTP and Met-tRNAi. When allowance is made for the limitation of diffusional coefficients imposed on macromolecules by the intracellular milieu it is apparent that recent estimates by Rowlands et al. (Eur. J. Biochem. 175, 93:1988) of higher concentrations of eIF-2 and eIF-2B in cells than hitherto proposed become necessary to support known rates of initiation. Under these conditions changing concentrations of met-tRNAi as proposed by Cooper and Braverman (J. Biol. Chem. 256, 7461:1981) are likely to have an important regulating influence.