Investigating the in vivo activity of the DeaD protein using protein–protein interactions and the translational activity of structured chloramphenicol acetyltransferase mRNAs

Abstract

Here, we report the use of an in vivo protein-protein interaction detection approach together with focused follow-up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow-up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow-up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large-scale protein-protein interaction data can be suitable to study protein functions in E. coli.

Base methylations in the double-stranded RNA by a fused methyltransferase bearing unwinding activity

“…However, disruption of the rlmKL gene showed almost no growth phenotype even when cells were cultured at low temperature (Figure 7A), and no significant change in ribosome profile was detected (Figure 7B). Since YcbY interacts with DeaD and SrmB in E. coli (45), we explored the synthetic growth phenotype of the Δ rlmKL strain in which RNA helicases, including deaD , srmB , rhlE and dbpA , were knocked out. As shown in Figure 7A, we observed a synthetic growth phenotype with deaD , in particular at low temperature.…”

RNA helicases

“…Peil et al 76 proposed that DeaD could affect r-protein translation leading to the lack of specific r-proteins in deaD mutants, as observed by Charollais et al 29 While DeaD has been associated with translaton, Peil et al 76 also show that the final activation step, as indicated by reduced peptide bond formation by the 40S particles, is affected in deaD mutants, suggesting a direct involvement of DeaD in ribosome assembly. DeaD interaction with a variety of nucleic acid binding proteins including large and small subunit ribosomal proteins, in addition to RNA and DNA methylases and RNase R in a non-RNA-dependent manner suggests that DeaD may perform additional roles in ribosome assembly and/or cellular metabolism in addition to alteration of rRNA secondary structure, 79 roles which are required at low temperature.…”

Global investigation of protein–protein interactions in yeast Saccharomyces cerevisiae using re-occurring short polypeptide sequences

“…Hence, 59 of the possible set of 300 novel interactions detected by PIPE2, or 20%, can also be supported by a functional relationship. Similarly, a second potential line of validation for an interaction might be that the interacting proteins often have common interactors (common third protein interaction) (10,30). Our manual survey of interaction databases from primary literature indicated that 49, 45 and 46 protein pairs among the three selected sets of novel interactions, respectively, had previously reported common interactions with at least one other protein (Table S6).…”