The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth

Plant photosynthesis and growth are often limited by the activity of the CO2-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco fromRhodobacter sphaeroides(RsRubisco)—ared-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimizedRs-rbcLSoperon, the messenger RNA (mRNA) abundance was ∼25% ofrbcLtranscript andRsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status ofRsRubisco in tobRsLS (∼23% sites active under ambient CO2), the metabolic repair proteinRsRca (Rs-activase) was introduced via nuclear transformation.RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m2) and enhancedRsRubisco activation to 75% under elevated CO2(1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producingRsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO2-fixation rates (kcatc) were reduced >95% and CO2/O2specificity impaired 30–50%. We show differences in hybrid and WTRsRubisco biogenesis in tobacco correlated with assembly inEscherichia coliadvocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineeredRsRubisco, an isoform amenable to directed evolution.

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Analysis of Messenger RNA Secondary Structures in Rhodobacter sphaeroides

10.21203/rs.3.rs-36110/v1 ◽

2020 ◽

Author(s):

Damilola Omotajo ◽

Hyuk Cho ◽

Madhusudan Choudhary

Keyword(s):

Secondary Structure ◽

Rhodobacter Sphaeroides ◽

Translation Initiation ◽

Messenger Rna ◽

Gc Content ◽

Secondary Structures ◽

Inhibitory Effect ◽

Nucleotide Composition ◽

Protein Coding ◽

Secondary Structure Analysis

Abstract Background: The Shine-Dalgarno (SD) sequence, when present, is known to promote translation initiation in a bacterial cell. However, the thermodynamic stability of the messenger RNA (mRNA) through its secondary structures has an inhibitory effect on the efficiency of translation. This poses the question of whether bacterial mRNAs with SD have low secondary structure formation or not. Results: About 3500 protein-coding genes in Rhodobacter sphaeroides were analyzed and a sliding window analysis of the last 100 nucleotides of the 5’ UTR and the first 100 nucleotides of ORFs was performed using RNAfold, a software for RNA secondary structure analysis. It was shown that mRNAs with SD are less stable than those without SD for genes located on the primary chromosome, but not for the plasmid encoded genes. Furthermore, mRNA stability is similar for genes within each chromosome except those encoded by the accessory chromosome (second chromosome). Conclusions: Results highlight the possible contribution of other factors like replicon- specific nucleotide composition (GC content), codon bias, and protein stability in determining the efficiency of translation initiation in both SD-dependent and SD-independent translation systems.

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Differential regulation of insulin-like growth factor binding protein-2 and -4 mRNA in muscle tissues and liver by diabetes or fasting

Journal of Endocrinology ◽

10.1677/joe.0.1430235 ◽

1994 ◽

Vol 143 (2) ◽

pp. 235-242 ◽

Cited By ~ 14

Author(s):

Y Chen ◽

H J Arnqvist

Keyword(s):

Skeletal Muscle ◽

Growth Factor ◽

Heart Muscle ◽

Messenger Rna ◽

Metabolic Regulation ◽

Mrna Levels ◽

Insulin Like Growth Factor ◽

Factor Binding ◽

Organ Specific ◽

Aortic Intima

Abstract The present study was undertaken to investigate the metabolic regulation of insulin-like growth factor binding proteins (IGFBPs) gene expression in muscles from diabetic or fasted rat. The messenger RNA (mRNA) levels for IGFBP-2 and -4 were analysed by solution hybridization in heart, skeletal and smooth muscle and liver from fasted (3 days) and refed (6, 12, 24, 72 h) rats and rats made diabetic with streptozotocin. In aortic intima-media, the mRNA levels for IGFBP-2 and -4 were decreased by diabetes or fasting and were restored gradually by refeeding. The response of IGFBP-4 mRNA to diabetes appeared two days after injection of streptozotocin, while a significant decrease of IGFBP-2 mRNA was found after a diabetes duration of two weeks. Both diabetes and fasting decreased IGFBP-4 mRNA levels in heart muscle and skeletal muscle and refeeding restored mRNA for IGFBP-4 to normal level. IGFBP-2 mRNA was undetectable in heart muscle and skeletal muscle. In liver IGFBP-4 mRNA was abundantly expressed. It was slightly but significantly decreased by fasting and approached normality with refeeding, while no change was found in diabetic liver. In contrast, liver IGFBP-2 mRNA was much lower in amount than IGF-I mRNA and IGFBP-4 mRNA and was sharply elevated by fasting, and decreased by refeeding. In conclusion, 1) both IGFBP-2 and -4 mRNA in various tissues are regulated by diabetes or fasting; 2) the mRNA for IGFBP-2 is metabolically regulated in a discordant, organ-specific manner. Journal of Endocrinology (1994) 143, 235–242

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The impact of a 48-h fast on SIRT1 and GCN5 in human skeletal muscle

Applied Physiology Nutrition and Metabolism ◽

10.1139/apnm-2016-0130 ◽

2016 ◽

Vol 41 (9) ◽

pp. 953-962 ◽

Cited By ~ 1

Author(s):

Brittany A. Edgett ◽

Trisha D. Scribbans ◽

James P. Raleigh ◽

Jennifer B.L. Matusiak ◽

Kristen Boonstra ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Messenger Rna ◽

Animal Studies ◽

Human Skeletal Muscle ◽

Muscle Protein ◽

Peak Oxygen Uptake ◽

The Impact ◽

Whole Muscle ◽

Activation Status

The present study examined the impact of a 48 h fast on the expression and activation status of SIRT1 and GCN5, the relationship between SIRT1/GCN5 and the gene expression of PGC-1α, and the PGC-1α target PDK4 in the skeletal muscle of 10 lean healthy men (age, 22.0 ± 1.5 years; peak oxygen uptake, 47.2 ± 6.7 mL/(min·kg)). Muscle biopsies and blood samples were collected 1 h postprandial (Fed) and following 48 h of fasting (Fasted). Plasma insulin (Fed, 80.8 ± 47.9 pmol/L; Fasted, not detected) and glucose (Fed, 4.36 ± 0.86; Fasted, 3.74 ± 0.25 mmol/L, p = 0.08) decreased, confirming participant adherence to fasting. Gene expression of PGC-1α decreased (p < 0.05, –24%), while SIRT1 and PDK4 increased (p < 0.05, +11% and +1023%, respectively), and GCN5 remained unchanged. No changes were observed for whole-muscle protein expression of SIRT1, GCN5, PGC-1α, or COX IV. Phosphorylation of SIRT1, AMPKα, ACC, p38 MAPK, and PKA substrates as well as nuclear acetylation status was also unaltered. Additionally, nuclear SIRT1 activity, GCN5, and PGC-1α content remained unchanged. Preliminary findings derived from regression analysis demonstrate that changes in nuclear GCN5 and SIRT1 activity/phosphorylation may contribute to the control of PGC-1α, but not PDK4, messenger RNA expression following fasting. Collectively, and in contrast with previous animal studies, our data are inconsistent with the altered activation status of SIRT1 and GCN5 in response to 48 h of fasting in human skeletal muscle.

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Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis

Journal of Bacteriology ◽

10.1128/jb.187.12.4222-4228.2005 ◽

2005 ◽

Vol 187 (12) ◽

pp. 4222-4228 ◽

Cited By ~ 16

Author(s):

Yehouda Marcus ◽

Hagit Altman-Gueta ◽

Aliza Finkler ◽

Michael Gurevitz

Keyword(s):

Binding Sites ◽

Phosphate Binding ◽

Bisphosphate Carboxylase ◽

Photoautotrophic Growth ◽

The Third ◽

Rate Of Photosynthesis ◽

Rubisco Activation ◽

Positively Charged ◽

Stimulation Of

ABSTRACT Orthophosphate (Pi) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds Pi at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the Pi-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the Pi inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the Km (RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas Pi binding only at the 5P site inhibits catalysis in a competitive manner.

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Probing green algal hydrogen production

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2002.1152 ◽

2002 ◽

Vol 357 (1426) ◽

pp. 1499-1509 ◽

Cited By ~ 51

Author(s):

Liping Zhang ◽

Anastasios Melis

Keyword(s):

Green Algae ◽

Metabolic Regulation ◽

Oxygenic Photosynthesis ◽

Oxygen Production ◽

Carbohydrate Accumulation ◽

Green Algal ◽

Water Rates ◽

Rate Of Photosynthesis ◽

First Time ◽

Regulation Of Photosynthesis

The recently developed two–stage photosynthesis and H 2 –production protocol with green algae is further investigated in this work. The method employs S deprivation as a tool for the metabolic regulation of photosynthesis. In the presence of S, green algae perform normal photosynthesis, carbohydrate accumulation and oxygen production. In the absence of S, normal photosynthesis stops and the algae slip into the H 2 –production mode. For the first time, to our knowledge, significant amounts of H 2 gas were generated, essentially from sunlight and water. Rates of H 2 production could be sustained continuously for ca . 80 h in the light, but gradually declined thereafter. This work examines biochemical and physiological aspects of this process in the absence or presence of limiting amounts of S nutrients. Moreover, the effects of salinity and of uncouplers of phosphorylation are investigated. It is shown that limiting levels of S can sustain intermediate levels of oxygenic photosynthesis, in essence raising the prospect of a calibration of the rate of photosynthesis by the S content in the growth medium of the algae. It is concluded that careful titration of the supply of S nutrients in the green alga medium might permit the development of a continuous H 2 –production process.

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High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166944 ◽

1996 ◽

Vol 54 ◽

pp. 896-897

Author(s):

G. W. Hacker ◽

I. Zehbe ◽

J. Hainfeld ◽

A.-H. Graf ◽

C. Hauser-Kronberger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Messenger Rna ◽

High Performance ◽

Detection System ◽

Direct Methods ◽

Genetic Alterations ◽

Chain Reaction ◽

Protein Encoding ◽

Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bcj20190674 ◽

2020 ◽

Vol 477 (1) ◽

pp. 173-189 ◽

Cited By ~ 1

Author(s):

Marco Pedretti ◽

Carolina Conter ◽

Paola Dominici ◽

Alessandra Astegno

Keyword(s):

Excision Repair ◽

Complex Structure ◽

Binding Motif ◽

C Terminus ◽

Repair Protein ◽

Nucleotide Excision ◽

Ef Hand ◽

Molecular Determinants ◽

Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

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