Comparative analysis of RNA regulatory elements of amino acid metabolism genes in Actinobacteria

Background  

Formation of alternative structures in mRNA in response to external stimuli, either direct or mediated by proteins or other RNAs, is a major mechanism of regulation of gene expression in bacteria. This mechanism has been studied in detail using experimental and computational approaches in proteobacteria and Firmicutes, but not in other groups of bacteria.     

The role of the small regulatory RNA GcvB in GcvB/mRNA posttranscriptional regulation of oppA and dppA in Escherichia coli

The gcvB gene encodes two small, nontranslated RNAs that regulate OppA and DppA, periplasmic binding proteins for the oligopeptide and dipeptide transport systems. Analysis of the gcvB sequence identified a region of complementarity near the ribosome-binding sites of dppA and oppA mRNAs. Several changes in gcvB predicted to reduce complementarity of GcvB with dppA-lacZ and oppA-phoA reduced the ability of GcvB to repress the target RNAs while other changes had no effect or resulted in stronger repression of the target mRNAs. Mutations in dppA-lacZ and oppA-phoA that restored complementarity to GcvB restored the ability of GcvB to repress dppA-lacZ but not oppA-phoA. Additionally, a change that reduced complementarity of GcvB to dppA-lacZ reduced GcvB repression of dppA-lacZ with no effect on oppA-phoA. The results suggest that different regions of GcvB have different roles in regulating dppA and oppA mRNA, and although pairing between GcvB and dppA mRNA is likely part of the regulatory mechanism, the results do not support a simple base pairing interaction between GcvB and its target mRNAs as the complete mechanism of repression.     

The small RNA Aar in Acinetobacter baylyi: a putative regulator of amino acid metabolism

Small non-coding RNAs (sRNAs) are key players in prokaryotic metabolic circuits, allowing the cell to adapt to changing environmental conditions. Regulatory interference by sRNAs in cellular metabolism is often facilitated by the Sm-like protein Hfq. A search for novel sRNAs in A. baylyi intergenic regions was performed by a biocomputational screening. One candidate, Aar, encoded between trpS and sucD showed Hfq dependency in Northern blot analysis. Aar was expressed strongly during stationary growth phase in minimal medium; in contrast, in complex medium, strongest expression was in the exponential growth phase. Whereas over-expression of Aar in trans did not affect bacterial growth, seven mRNA targets predicted by two in silico approaches were upregulated in stationary growth phase. All seven mRNAs are involved in A. baylyi amino acid metabolism. A putative binding site for Lrp, the global regulator of branched-chain amino acids in E. coli, was observed within the aar gene. Both facts imply an Aar participation in amino acid metabolism.     

Reducing plasma HIV RNA improves muscle amino acid metabolism

We reported (Yarasheski KE, Zachwieja JJ, Gischler J, Crowley J, Horgan MM, and Powderly WG. Am J Physiol Endocrinol Metab 275: E577-E583, 1998) that AIDS muscle wasting was associated with an inappropriately low rate of muscle protein synthesis and an elevated glutamine rate of appearance (Ra Gln). We hypothesized that high plasma HIV RNA caused dysregulation of muscle amino acid metabolism. We determined whether a reduction in HIV RNA (> or =1 log) increased muscle protein synthesis rate and reduced R(a) Gln and muscle proteasome activity in 10 men and 1 woman (22-57 yr, 60-108 kg, 17-33 kg muscle) with advanced HIV (CD4 = 0-311 cells/microl; HIV RNA = 10-375 x 10(3) copies/ml). We utilized stable isotope tracer methodologies ([13C]Leu and [15N]Gln) to measure the fractional rate of mixed muscle protein synthesis and plasma Ra Gln in these subjects before and 4 mo after initiating their first or a salvage antiretroviral therapy regimen. After treatment, median CD4 increased (98 vs. 139 cells/microl, P = 0.009) and median HIV RNA was reduced (155,828 vs. 100 copies/ml, P = 0.003). Mixed muscle protein synthesis rate increased (0.062 +/- 0.005 vs. 0.078 +/- 0.006%/h, P = 0.01), Ra Gln decreased (387 +/- 33 vs. 323 +/- 15 micromol.kg fat-free mass(-1).h(-1), P = 0.04), and muscle proteasome chymotrypsin-like catalytic activity was reduced 14% (P = 0.03). Muscle mass was only modestly increased (1 kg, P = not significant). We estimated that, for each 10,000 copies/ml reduction in HIV RNA, approximately 3 g of additional muscle protein are synthesized per day. These findings suggest that reducing HIV RNA increases muscle protein synthesis and reduces muscle proteolysis, but muscle protein synthesis relative to whole body protein synthesis rate is not restored to normal, so muscle mass is not substantially increased.     

Glucocorticoid regulation of amino acid and polyamine metabolism in the small intestine

Several factors (including diets, changes in intestinal fluora, and hormones) regulate postnatal intestinal growth and development. Based on the early studies involving modification of the adrenal gland, pituitary gland or hypothalamus, exogenous glucocorticoids and glucocorticoid receptor antagonists are now used to study glucocorticoid-mediated metabolism of amino acids in the small intestine. Findings from these studies indicate that physiological levels of glucocorticoids stimulate the catabolism of glutamine and proline for the synthesis of citrulline and arginine in enterocytes during weaning. In addition, increases in circulating levels of glucocorticoids enhance expression of arginase, proline oxidase and ornithine decarboxylase, as well as polyamine synthesis from arginine and proline in enterocytes. These actions of the hormones promote intestinal maturation and may have therapeutic effects on intestinal disease (e.g., necrotizing enterocolitis). Molecular aspects, species-specific effects, and developmental responsiveness to glucocorticoids should be taken into consideration in designing both experimental and clinical studies.     

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA-RNA interactions.     

CpdA is involved in amino acid metabolism in Shewanella oneidensis MR-1

Cyclic 3′,5′-adenosine monophosphate (cAMP) phosphodiesterase (CPD) is an enzyme that catalyzes the hydrolysis of cAMP, a signaling molecule affecting diverse cellular and metabolic processes in bacteria. Some CPDs are also known to function in cAMP-independent manners, while their physiological roles remain largely unknown. Here, we investigated physiological roles of CPD in Shewanella oneidensis MR-1, a model environmental bacterium, and report that CPD is involved in amino-acid metabolism. We found that a CPD-deficient mutant of MR-1 (ΔcpdA) showed decreased expression of genes for the synthesis of methionine, S-adenosylmethionine, and histidine and required these three compounds to grow in minimal media. Interestingly, deletion of adenylate cyclases in ΔcpdA did not restore the ability to grow in minimal media, indicating that the amino acid requirements were not due to the accumulation of cAMP. These results suggest that CPD is involved in the regulation of amino acid metabolism in MR-1 in a cAMP-independent manner.     

Allosteric mechanisms in ACT domain containing enzymes involved in amino acid metabolism

Summary. An important sequence motif identified by sequence analysis is shared by the ACT domain family, which has been found in a number of diverse proteins. Most of the proteins containing the ACT domain seem to be involved in amino acid and purine synthesis and are in many cases allosteric enzymes with complex regulation enforced by the binding of ligands. Here we explore the current understanding of the ACT domain function including its role as an allosteric module in a selected group of enzymes. We will further describe in more detail three of the proteins where some understanding is available on function and structure: i) the archetypical ACT domain protein E. coli 3PGDH, which catalyzes the first step in the biosynthesis of L-Ser, ii) the bifunctional chorismate mutase/prephenate dehydratase (P-protein) from E. coli, which catalyzes the first two steps in the biosynthesis of L-Phe, and iii) the mammalian aromatic amino acid hydroxylases, with special emphasis on phenylalanine hydroxylase, which catalyzes the first step in the catabolic degradation of L-phenylalanine (L-Phe). The ACT domain is commonly involved in the binding of a small regulatory molecule, such as the amino acids L-Ser and L-Phe in the case of 3PGDH and P-protein, respectively. On the other hand, for PAH, and probably for other enzymes, this domain appears to have been incorporated as a handy, flexible small module with the potential to provide allosteric regulation via transmission of finely tuned conformational changes, not necessarily initiated by regulatory ligand binding at the domain itself.Current address: Protein Biophysics & Delivery, Novo Nordisk A/S, Novo Allé, 2880 Bagsværd, Denmark.     

Highly conserved protein kinases involved in the regulation of carbon and amino acid metabolism

It has been clear for over a decade and a half that ancient signalling pathways controlling fundamental cellular processes are highly conserved throughout the eukaryotes. Two plant protein kinases, sucrose non-fermenting 1 (SNF1)-related protein kinase (SnRK1) and general control non-derepressible 2 (GCN2)-related protein kinase are reviewed here. These protein kinases show an extraordinary level of conservation with their fungal and animal homologues given the span of time since they diverged from them. However, close examination of the signalling pathways in which they operate also reveals intriguing differences in activation and function.