Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains.

NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochrome P450 and catalyzes the one-electron reduction of many drugs and foreign compounds. This enzyme is a flavoprotein containing the cofactors FMN and FAD, which are essential for its function. We have expressed the putative FMN and FAD/NADPH binding domains of P450 reductase and show that these distinct peptides fold correctly to bind their respective cofactors. The FAD/NADPH domain catalyzed the one-electron reduction of a variety of substrates but did not efficiently reduce cytochrome c or cytochrome P450 (as judged by the oxidation of the CYP1A1 substrate 7-ethoxyresorufin). However, the domains could be combined to provide a functional enzyme active in the reduction of cytochrome c and in transferring electrons to cytochrome P450. Both the reconstitution of the domains and the direct binding of cytochrome c to the FMN domain were ionic-strength dependent. The FMN domain containing the hydrophobic membrane anchor sequence was a potent inhibitor of reconstituted monooxygenase activity. These data strongly support the hypothesis that FMN/FAD-containing proteins have evolved as a fusion of two ancestral genes and provide fundamental insights into how this and structurally related proteins, such as nitric oxide synthase and sulfite reductase, have evolved and function.     

Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis.

Linker-insertion mutations were introduced into the cloned adenovirus DNA polymerase gene and the functional effects on the initiation and elongation of DNA in vitro were measured. Essential regions of the polymerase appear to be scattered in patches across the entire molecule and are not limited to the five regions of homology shared with a variety of other replicating polymerases. Thus, the adenovirus DNA polymerase presumably contains active sites that must be formed by distant interactions across the polymerase molecule.     

ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli. Mutant proteins with alanine at position 66 replaced by glycine [eIF-4A(A66G)] or valine [eIF-4A(A66V)] were purified from Escherichia coli extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays. The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor. Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays. In vitro translation was supported by wild-type eIF-4A and eIF-4A(A66G) but not by eIF-4A(A66V). The results show that, for their translation, the majority of mRNAs from Saccharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.     

Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA.

Translation of the genetic code requires the accurate selection of elongation factor (EF)-Tu.GTP.tRNA ternary complexes at the ribosomal acceptor site, or A site. Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified. Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G-->A substitution at position 530. We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se. In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex. These results provide evidence for a newly defined function of 16S rRNA--namely, modulation of EF-Tu activity during translation.     

Eukaryotic initiation factor 3 is required for poliovirus 2A protease-induced cleavage of the p220 component of eukaryotic initiation factor 4F.

After cultured cells are infected with poliovirus, cellular mRNA fails to bind to ribosomes, and synthesis of the majority of cellular proteins ceases. The defective step has been localized to the cap-dependent activity of the eukaryotic translation initiation factor 4F. Inactivation of this factor correlates with the cleavage of its largest subunit, p220, into characteristic products observed in infected cells. This cleavage is mediated by the poliovirus protease 2Apro. Previous work suggests that 2Apro does not catalyze the reaction directly, suggesting that one or more cellular proteins is required for the degradation of p220. To identify such a protein, we have developed an assay in which cleavage of a p220 substrate in the presence of poliovirus 2Apro is dependent upon the addition of HeLa cell proteins. By using this assay, we show that another factor, eukaryotic translation initiation factor 3, is required for 2Apro-dependent cleavage of p220.