Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases, Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of Prp2, an N-terminally truncated construct of Chaetomium thermophilum Prp2 was crystallized in the presence of ADP-BeF3 − and a poly-U12 RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF3 −- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However, Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3′ stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.

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The Gram-positive model bacterium Bacillus subtilis is able to utilize a variety of proteinogenic and non-proteinogenic amino acids as sources of carbon, energy and nitrogen. The utilization of the amino acids arginine, citrulline and ornithine is catalyzed by enzymes that are encoded in the rocABC and rocDEF operons and by the rocG gene. Expression of these genes is under control of the alternative sigma factor SigL. RNA polymerase associated to this sigma factor depends on an ATP-hydrolyzing transcription activator to initiate transcription. The RocR protein acts as transcription activator for the roc genes. In this work, we have studied the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline and ornithine. This identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that it acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to ornithine and carbamate. So far, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments we demonstrate that ornithine is the molecular inducer for RocR activity. Our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector for the transcription regulator RocR.

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