The rnf genes of Rhodobacter capsulatus, essential for nitrogen fixation, are thought to encode a system for electron transport to nitrogenase. In the present study, we have attempted to overexpress the rnf genes in Escherichia coli to investigate the molecular properties of the corresponding proteins. Corrections were made to the published DNA sequence of the rnf operon, resulting in the identification of two genes, rnfG and rnfH. The rnfABCDGEH operon thus comprises seven genes and shows similarities in gene arrangement and deduced protein sequences to homologous regions in the genomes of Haemophilus influenzae and E. coli. Four of the rnf gene products were found to be similar in sequence to components of an Na ϩ -dependent NADH:ubiquinone oxidoreductase from Vibrio alginolyticus. Three of the rnf genes were successfully overexpressed in E. coli as His-tagged polypeptides, whereas the products of rnfA, rnfD and rnfE, predicted to be transmembrane proteins, could not be stably maintained in E. coli. The rnfB and rnfC gene products were isolated as two brown proteins with apparent molecular-mass values of 25 kDa and 55 kDa, respectively. RnfB was shown to contain one [2Fe-2S] cluster, based on absorption spectrophotometry, EPR spectroscopy and iron content. Recombinant RnfC contained at least one ironsulfur cluster, most likely of the [4Fe-4S] type. Unambiguous identification of the prosthetic groups was, however, precluded by the extreme instability of this protein. In R. capsulatus, RnfB and RnfC were found by immunoblot analysis to be tightly bound to the membrane, despite their hydrophilic character. The RnfB and RnfC proteins were absent in mutant strains bearing insertions at various positions within the rnfABCDGEH operon, suggesting that their stability depends on the cosynthesis of the other rnf gene products. We observed that iron limitation during growth resulted in a decrease both in the cellular content of RnfB and in the level of transcription of the rnfABCDGEH operon, indicating that the expression of this operon is regulated as a function of iron availability

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA.