RESUMO
Biological carbon dioxide fixation is an essential and crucial process catalyzed by both prokaryotic and eukaryotic organisms to allow ubiquitous atmospheric CO2 to be reduced to usable forms of organic carbon. This process, especially the Calvin-Bassham-Benson (CBB) pathway of CO2 fixation, provides the bulk of organic carbon found on earth. The enzyme ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) performs the key and rate-limiting step whereby CO2 is reduced and incorporated into a precursor organic metabolite. This is a highly regulated process in diverse organisms, with the expression of genes that comprise the CBB pathway (the cbb genes), including RubisCO, specifically controlled by the master transcriptional regulator protein CbbR. Many organisms have two or more cbb operons that either are regulated by a single CbbR or employ a specific CbbR for each cbb operon. CbbR family members are versatile and accommodate and bind many different effector metabolites that influence CbbR's ability to control cbb transcription. Moreover, two members of the CbbR family are further posttranslationally modified via interactions with other transcriptional regulator proteins from two-component regulatory systems, thus augmenting CbbR-dependent control and optimizing expression of specific cbb operons. In addition to interactions with small effector metabolites and other regulator proteins, CbbR proteins may be selected that are constitutively active and, in some instances, elevate the level of cbb expression relative to wild-type CbbR. Optimizing CbbR-dependent control is an important consideration for potentially using microbes to convert CO2 to useful bioproducts.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo do Carbono/fisiologia , Dióxido de Carbono/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/genéticaRESUMO
The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K(+) channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function.
Assuntos
Cerebelo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Canais de Potássio Shaw/metabolismo , Animais , Células Cultivadas , Cerebelo/citologia , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Ratos , Canais de Potássio Shaw/genética , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
CbbR is a LysR-type transcriptional regulator that activates expression of the operons containing (cbb) genes that encode the CO2 fixation pathway enzymes in Ralstonia eutropha (Cupriavidus necator) under autotrophic growth conditions. The cbb operons are stringently downregulated during chemoheterotrophic growth on organic acids such as malate. CbbR constitutive proteins (CbbR*s), typically with single amino acid substitutions, were selected and isolated that activate expression of the cbb operons under chemoheterotrophic growth conditions. A large set of CbbR*s from all major domains of the CbbR molecule were identified, except for the DNA-binding domain. The level of gene expression conferred for many of these CbbR*s under autotrophic growth was greater than that conferred by wild-type CbbR. Several of these CbbR*s increase transcription two- to threefold more than wild-type CbbR. One particular CbbR*, a truncated protein, was useful in identifying the regions of CbbR that are necessary for transcriptional activation and, by logical extension, necessary for interaction with RNA polymerase. The reductive assimilation of carbon via CO2 fixation is an important step in the cost-effective production of useful biological compounds. Enhancing CO2 fixation in Ralstonia eutropha through greater transcriptional activation of the cbb operons could prove advantageous, and the use of CbbR*s is one way to enhance product formation.
Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas de Ligação a DNA/química , Expressão Gênica , Genes Reporter , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
CbbR and RegA (PrrA) are transcriptional regulators of the Calvin-Benson-Bassham (CBB) CO2 fixation pathway (cbbI and cbbII) operons of Rhodobacter sphaeroides. The CbbR and RegA proteins interact, but CbbR must be bound to the promoter DNA in order for RegA-CbbR protein-protein interactions to occur. RegA greatly enhances the ability of CbbR to bind the cbbI promoter or greatly enhances the stability of the CbbR/promoter complex. The N-terminal receiver domain and the DNA binding domain of RegA were shown to interact with CbbR. Residues in α-helix 7 and α-helix 8 of the DNA binding domain (helix-turn-helix) of RegA directly interacted with CbbR, with α-helix 7 positioned immediately above the DNA and α-helix 8 located in the major groove of the DNA. A CbbR protein containing only the DNA binding motif and the linker helix was capable of binding to RegA. In contrast, a truncated CbbR containing only the linker helix and recognition domains I and II (required for effector binding) was not able to interact with RegA. The accumulated results strongly suggest that the DNA binding domains of both proteins interact to facilitate optimal transcriptional control over the cbb operons. In vivo analysis, using constitutively active mutant CbbR proteins, further indicated that CbbR must interact with phosphorylated RegA in order to accomplish transcriptional activation.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Rhodobacter sphaeroides/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Mutação , Ligação Proteica , Conformação Proteica , Rhodobacter sphaeroides/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
CbbR and RegA (PrrA) are transcriptional regulators of the cbb(I) and cbb(II) (Calvin-Benson-Bassham CO(2) fixation pathway) operons of Rhodobacter sphaeroides. Both proteins interact specifically with promoter sequences of the cbb operons. RegA has four DNA binding sites within the cbb(I) promoter region, with the CbbR binding site and RegA binding site 1 overlapping each other. This study demonstrated that CbbR and RegA interact and form a discrete complex in vitro, as illustrated by gel mobility shift experiments, direct isolation of the proteins from DNA complexes, and chemical cross-linking analyses. For CbbR/RegA interactions to occur, CbbR must be bound to the DNA, with the ability of CbbR to bind the cbb(I) promoter enhanced by RegA. Conversely, interactions with CbbR did not require RegA to bind the cbb(I) promoter. RegA itself formed incrementally larger multimeric complexes with DNA as the concentration of RegA increased. The presence of RegA binding sites 1, 2 and 3 promoted RegA/DNA binding at significantly lower concentrations of RegA than when RegA binding site 3 was not present in the cbb(I) promoter. These studies support the premise that both CbbR and RegA are necessary for optimal transcription of the cbb(I) operon genes of R. sphaeroides.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Rhodobacter sphaeroides/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Rhodobacter sphaeroides/genética , Transativadores/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Nonsulfur purple (NSP) photosynthetic bacteria use the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway for the reduction of CO(2) via ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (RubisCO), as a means to build cell mass during chemoautotrophic or photoautotrophic conditions. In addition, the CBB pathway plays an important role in maintaining redox balance during photoheterotrophic growth conditions. In this communication we describe protein-protein interactions between two transcriptional regulators CbbR and RegA and the possible role of the CbbX protein in regulating the CBB pathway in Rhodobacter sphaeroides. In Rhodopseudomonas palustris, the CbbR and the CbbRRS system (a three-protein, two-component regulatory system) regulate the CBB pathway. Moreover, derepression of the nitrogenase complex, and the production of hydrogen gas, appears to be a common mechanism to balance the redox potential in RubisCO-compromised strains of NSP photosynthetic bacteria.
Assuntos
Carbono/metabolismo , Hidrogênio/metabolismo , Nitrogênio/metabolismo , Fotossíntese/fisiologia , Rhodobacter sphaeroides/metabolismo , Enxofre/metabolismo , Rhodobacter sphaeroides/genéticaRESUMO
CbbR is a LysR-type transcriptional regulator (LTTR) that is required to activate transcription of the cbb operons, responsible for CO2 fixation, in Rhodobacter sphaeroides. LTTR proteins often require a co-inducer to regulate transcription. Previous studies suggested that ribulose 1,5-bisphosphate (RuBP) is a positive effector for CbbR function in this organism. In the current study, RuBP was found to increase the electrophoretic mobility of the CbbR/cbb(I) promoter complex. To define and analyse the co-inducer recognition region of CbbR, constitutively active mutant CbbR proteins were isolated. Under growth conditions that normally maintain transcriptionally inactive cbb operons, the mutant CbbR proteins activated transcription. Fourteen of the constitutively active mutants resulted from a single amino acid substitution. One mutant was derived from amino acid substitutions at two separate residues that appeared to act synergistically. Different mutant proteins showed both sensitivity and insensitivity to RuBP and residues that conferred constitutive transcriptional activity could be highlighted on a three-dimensional model, with several residues unique to CbbR shown to be at locations critical to LTTR function. Many of the constitutive residues clustered in or near two specific loops in the LTTR tertiary structure, corresponding to a proposed site of co-inducer binding.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Resistência a Medicamentos/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter/genética , Mutação , Óperon/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Ribulosefosfatos/farmacologia , Fatores de Transcrição/genética , Transcrição Gênica , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
The 5' long terminal repeat of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of intron-containing human immunodeficiency virus type 1 (HIV-1) gag. The SNV RU5 region, which corresponds to the 165-nucleotide 5' RNA terminus, functions in a position- and orientation-dependent manner to enhance polysome association of intron-containing HIV-1 gag RNA and also nonviral luc RNA. Evidence is mounting that association with nuclear factors during intron removal licenses mRNAs for nuclear export, efficient translation, and nonsense-mediated decay. This project addressed the relationship between the nuclear export pathway of SNV RU5-reporter RNA and translational enhancement. Results of RNA transfection experiments suggest that cytoplasmic proteins are insufficient for SNV RU5 translational enhancement of gag or luc RNA. Reporter gene assays, leptomycin B (LMB) sensitivity experiments, and RNase protection assays indicate that RU5 gag RNA accesses a nuclear export pathway that is distinct from the LMB-inhibited leucine-rich nuclear export sequence-dependent CRM1 pathway, which is used by the HIV-1 RRE. As a unique tool with which to investigate the relationship between different RNA trafficking routes and translational enhancement, SNV RU5 and Rev/RRE were combined on a single gag RNA. We observed a less-than-synergistic effect on cytoplasmic mRNA utilization. Instead, Rev/RRE diverts RU5 gag RNA to the CRM1-dependent, LMB-inhibited pathway and abrogates translational enhancement by SNV RU5. Our study is the first to show that a nuclear factor(s) directs SNV RU5-containing RNAs to a distinct export pathway that is not inhibited by LMB and programs the intron-containing transcript for translational enhancement.