Wael Abdel-Fattah

The PhoP–PhoR two-component system (TCS) controls transcription of the Pho regulon in response to inorganic phosphate limitation in Bacillus subtilis. In addition to its role in phosphate homeostasis, increasing evidence suggests that central metabolic pathways influence Pho regulon activity. Here, we investigated the mechanism underlying phosphate-independent activation of the Pho regulon in a ccpA mutant lacking the global regulator of CCR. Gene expression analyses demonstrated strong glucose-dependent induction of Pho regulon genes in the absence of both CcpA and the cognate histidine kinase PhoR. Using PhosTag gel electrophoresis analysis and a phoasphoablative mutation in phoP (D53A), we detected early PhoP phosphorylation and determined that Pho regulon activation requires a phosphorylatable form of PhoP, even when the cognate histidine kinase phoR gene was deleted. Because PhoR can phosphorylate non-cognate response regulator YycF, we examined whether the essential YycFG signaling system contributes to PhoP activation. Co-immunoprecipitation and in vitro phosphorylation assays confirmed a direct interaction between PhoP and YycG and demonstrated in vitro PhoP phosphorylation by YycG. Taken together, we propose a model in which CcpA limits excessive PhoP accumulation to preserve signaling specificity and prevent aberrant activation through non-cognate kinases. Furthermore, our study reveals a previously unrecognized link between carbon catabolite repression and phosphate starvation signaling and demonstrates another crosstalk between two-component signal transduction systems in the Gram-positive bacterium, B. subtilis.

The PhoP-PhoR two-component system regulates transcription of the Pho regulon to counteract inorganic phosphate (Pi) limitation. Interestingly, in a ccpA mutant lacking the global regulator of carbon catabolism, Pho regulon genes are hyper-induced in a PhoR-independent, glucose-dependent manner. While CcpA is known to directly repress the phoP promoter, we now show that overexpression of a phosphorylatable form of PhoP is essential for this hyper-induction. Remarkably, we detected early PhoP phosphorylation in the ccpA phoR double mutant. PhosTag-based analysis revealed that the phosphorylated/unphosphorylated PhoP ratio, rather than absolute levels, is the critical determinant of Pho regulon activation under these conditions. Given that PhoR can phosphorylate non-cognate YycF, the response regulator of the essential YycFG system controlling cell wall metabolism. We hypothesized that PhoP phosphorylation in the absence of PhoR may be mediated by YycG. Supporting this, PhoP co- immunoprecipitated with YycG, and in vitro phosphorylation assays confirmed direct phosphorylation of PhoP by YycG. We propose a Pi-stress regulatory model in which CcpA prevents excessive PhoP accumulation, thereby maintaining an appropriate balance between phosphorylated and unphosphorylated PhoP. This regulatory balance prevents aberrant PhoP activation via non-cognate kinases such as YycG in the absence of PhoR. Our findings highlight the interplay between carbon catabolite repression and phosphate starvation responses, and reveal a novel mechanism by which non- cognate phosphorylation contributes to adaptive transcriptional regulation.

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2–8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3′-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3′-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron–exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.

Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure and function of its regulatory factor Kti12 have remained elusive. Here, we present the crystal structure of Kti12′s nucleotide hydrolase domain trapped in a transition state of ATP hydrolysis. The structure reveals striking similarities to an O-phosphoseryl-tRNA kinase involved in the selenocysteine pathway. Both proteins employ similar mechanisms of tRNA binding and show tRNA^Sec-dependent ATPase activity. In addition, we demonstrate that Kti12 binds directly to Elongator and that ATP hydrolysis is crucial for Elongator to maintain proper tRNA anticodon modification levels in vivo. In summary, our data reveal a hitherto uncharacterized link between two translational control pathways that regulate selenocysteine incorporation and affect ribosomal tRNA selection via specific tRNA modifications.

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin’s action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g., nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin’s action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g. nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.

Sang Eun Jun, Kiu-Hyung Cho, Ji-Young Hwang, Wael Abdel-Fattah, Alexander Hammermeister, Raffael Schaffrath, John L. Bowman, and Gyung-Tae Kim. Mol. Cells 2015;38:243-50. https://doi.org/10.14348/molcells.2015.2297

Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase. tRNA molecules function as adapters in protein synthesis, bringing amino acids to the ribosome and reading the genetic code through codon-anticodon base pairing. When the tRNA contains a uridine residue in the “wobble position” of its anticodon, which base-pairs with purine residues in the third position of a cognate codon, it is almost always chemically modified and modification is required for efficient decoding. In eukaryotic cells, these wobble uridine modifications require a conserved protein complex called Elongator. Our work shows that Elp1, Elongator's largest subunit, is phosphorylated on several sites. By blocking phosphorylation at these positions using mutations, we identified four phosphorylation sites that are important for Elongator's role in tRNA modification. We have also shown that Hrr25 protein kinase, a member of the casein kinase I (CKI) family, is responsible for modification of two of the sites that are important for Elongator function. Phosphorylation appears to affect interaction of the Elongator complex both with its kinase (Hrr25) and with Kti12, an accessory protein previously implicated in Elongator function. Our studies imply that Elp1 phosphorylation plays a positive role in Elongator-mediated tRNA modification and raise the possibility that wobble uridine modification may be regulated, representing a potential translational control mechanism.

Summary: Diphthamide is a conserved modification in archaeal and eukaryal translation elongation factor 2 (EF2). Its name refers to the target function for diphtheria toxin, the disease‐causing agent that, through ADP ribosylation of diphthamide, causes irreversible inactivation of EF2 and cell death. Although this clearly emphasizes a pathobiological role for diphthamide, its physiological function is unclear, and precisely why cells need EF2 to contain diphthamide is hardly understood. Nonetheless, the conservation of diphthamide biosynthesis together with syndromes (i.e. ribosomal frame‐shifting, embryonic lethality, neurodegeneration and cancer) typical of mutant cells that cannot make it strongly suggests that diphthamide‐modified EF2 occupies an important and translation‐related role in cell proliferation and development. Whether this is structural and/or regulatory remains to be seen. However, recent progress in dissecting the diphthamide gene network (DPH1–DPH7) from the budding yeast Saccharomyces cerevisiae has significantly advanced our understanding of the mechanisms required to initiate and complete diphthamide synthesis on EF2. Here, we review recent developments in the field that not only have provided novel, previously overlooked and unexpected insights into the pathway and the biochemical players required for diphthamide synthesis but also are likely to foster innovative studies into the potential regulation of diphthamide, and importantly, its ill‐defined biological role.

Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor. Intriguingly, dph1 truncation mutants are sensitive to overexpression of DPH5, the gene necessary to synthesize diphthine from the first diphthamide pathway intermediate produced by Dph1-Dph3. This is in stark contrast to dph6 mutants, which also lack the ability to form diphthamide but are resistant to growth inhibition by excess Dph5 levels. As judged from site-specific mutagenesis, the amidation reaction itself relies on a conserved ATP binding domain in Dph6 that, when altered, blocks diphthamide formation and confers resistance to eEF2 inhibition by sordarin.