L-myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the rate-limiting step in the synthesis of myo-inositol, a critical compound in the cell. Plants contain multiple MIPS genes, which encode highly similar enzymes. We characterized the expression patterns of the three MIPS genes in Arabidopsis thaliana and found that MIPS1 is expressed in most cell types and developmental stages, while MIPS2 and MIPS3 are mainly restricted to vascular or related tissues. MIPS1, but not MIPS2 or MIPS3, is required for seed development, for physiological responses to salt and abscisic acid, and to suppress cell death. Specifically, a loss in MIPS1 resulted in smaller plants with curly leaves and spontaneous production of lesions. The mips1 mutants have lower myo-inositol, ascorbic acid, and phosphatidylinositol levels, while basal levels of inositol (1,4,5)P 3 are not altered in mips1 mutants. Furthermore, mips1 mutants exhibited elevated levels of ceramides, sphingolipid precursors associated with cell death, and were complemented by a MIPS1-green fluorescent protein (GFP) fusion construct. MIPS1-, MIPS2-, and MIPS3-GFP each localized to the cytoplasm. Thus, MIPS1 has a significant impact on myo-inositol levels that is critical for maintaining levels of ascorbic acid, phosphatidylinositol, and ceramides that regulate growth, development, and cell death.

The rates of RNA synthesis and nascent RNA folding into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA exit channel can increase pausing by interacting with bacterial RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain is proposed to mediate some effects of nascent RNA structures. However, the connections among RNA structure formation, clamp movement, and catalytic activity remain uncertain. We assayed exit-channel structure formation in Escherichia coli RNAP together with disulfide crosslinks that favor closed or open clamp conformations and found that clamp position directly influences RNA structure formation and catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening and through interactions with the flap that slow translocation.

Translocation of RNA polymerase on DNA is thought to involve oscillations between pretranslocated and posttranslocated states that are rectified by nucleotide addition or pyrophosphorolysis. The pretranslocated register is also a precursor to transcriptional pause states that mediate regulation of transcript elongation. However, the determinants of bias between the pretranslocated and posttranslocated states are incompletely understood. To investigate translocation bias in multisubunit RNA polymerases, we measured rates of pyrophosphorolysis, which occurs in the pretranslocated register, in minimal elongation complexes containing T. thermophilus or E. coli RNA polymerase. Our results suggest that the identity of RNA:DNA nucleotides in the active site are strong determinants of susceptibility to pyrophosphorolysis, and thus translocation bias, with the 3′ RNA nucleotide favoring the pretranslocated state in the order U > C > A > G. The preference of 3′ U vs G for the pretranslocated register appeared to be universal among both bacterial and eukaryotic RNA polymerases and was confirmed by exonuclease III footprinting of defined elongation complexes. However, the relationship of pyrophosphate concentration to the rate of pyrophosphorolysis of 3′ U- versus 3′ G-containing elongation complexes did not match predictions of a simple mechanism in which 3′-RNA seqeunce affects only translocation bias and pyrophosphate (PPi) binds only to the pretranslocated state.

Background:In bacterial transcription complexes, nascent RNA structures increase pause duration. Results: Antisense RNA and to a lesser extent DNA oligonucleotides mimic effects of RNA structures synergistically with NusA and competitively with RfaH. Conclusion: Exit channel duplexes interact specifically with RNA polymerase and NusA and compete with RfaH to control clamp position. Significance: Insights into conformational dynamics of RNA polymerase improve mechanistic understanding of transcriptional regulation.

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.