Regulation of expression of photosynthesis genes in anoxygenic photosynthetic bacteria.
Explore the source record for details and available documents.
Base Sequence↗
Biomedical subjects
G Klug
Publications and source records attributed to G Klug.
Identification of a gene required for the oxygen-regulated formation of the photosynthetic apparatus of Rhodobacter capsulatus.
The pigment-binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild-type strain. The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension. The formation of wild-type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R. capsulatus wild-type chromosome or by addition of 10 micrograms l-1 vitamin B12 to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7 kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.
Amino Acid Sequence↗
The role of mRNA degradation in the regulated expression of bacterial photosynthesis genes.
Regulation of gene expression in bacteria, as in eukaryotic cells, is often achieved by variation of mRNA levels. Since the steady state levels of mRNA depend on both the rate of synthesis and the rate of decay, both mechanisms are important for gene regulation. After considerable effort undertaken over many years to understand the regulation of transcription, mRNA degradation has recently gained increasing attention as an important step in the regulation of some bacterial genes, and many investigations have addressed the mechanisms involved in mRNA decay. The puf mRNA of Rhodobacter capsulatus encoding pigment binding proteins has become a model system to study decay of a polycistronic mRNA and the role of mRNA degradation in gene expression. Individual segments of the polycistronic puf mRNA display extremely different half-lives. These differences in stability of mRNA segments are involved in the differential expression of puf encoded genes and consequently contribute to the stoichiometry of light-harvesting I and reaction centre complexes that results in optimal growth. In addition, control of mRNA stability is involved in the oxygen-dependent regulation of photosynthesis genes. High oxygen tension results in decreased stability of the reaction-centre specific puf mRNA segment, most likely by affecting the rate of endonucleolytic cleavage within the reaction centre coding region. The results obtained from studying puf mRNA degradation in R. capsulatus and Escherichia coli suggest that a specific distribution of decay promoting and decay impeding mRNA elements along the polycistronic mRNA is responsible for the different half-lives of individual puf segments.
Bacterial Proteins↗
The rate of decay of Rhodobacter capsulatus-specific puf mRNA segments is differentially affected by RNase E activity in Escherichia coli.
In Rhodobacter capsulatus the puf operon encodes proteins of the photosynthetic apparatus. The polycistronic puf mRNA is comprised of segments that show differential stability. Here, we show that the rate of decay of the 2.7-kb pufBALMX mRNA species in Escherichia coli depends on the activity of ribonuclease E (RNase E), whereas the degradation of the 0.5-kb pufBA mRNA segment is not affected by a mutation in the rne gene. The RNase E-promoted decay of the pufLMX mRNA depends on the presence of a 1.4-kb pufLM mRNA segment, in which rate-limiting endonucleolytic cleavage was postulated to occur in R. capsulatus. The insertion of 185 bp of this 1.4-kb segment into pufB results in an RNase E-dependent decay of the modified pufBA mRNA segment in E. coli. Our findings suggest that in R. capsulatus an RNase E-like activity is responsible for the rate-limiting endonucleolytic cleavage occurring within the pufLM mRNA segment, whereas the 0.5-kb pufBA mRNA segment is degraded by a different RNase E-independent decay mechanism.
Blotting, Northern↗
Endonucleolytic degradation of puf mRNA in Rhodobacter capsulatus is influenced by oxygen.
The formation of pigment-protein complexes in facultatively photosynthetic bacteria is regulated by the oxygen tension in the culture. It is shown that the degradation of some mRNA species encoding components of the photosynthetic apparatus is affected by oxygen. The puf mRNA segment, encoding the pigment-binding proteins of the reaction center, and the 0.5-kb puc mRNA species, encoding pigment-binding proteins of the light-harvesting LHII antenna complex of Rhodobacter capsulatus were degraded more rapidly under high oxygen tension than under low oxygen tension. Studies on strains having deletions or insertions in the puf operon indicate that rate-limiting endonucleolytic cleavage in the reaction center coding region of the polycistronic puf mRNA was influenced by growth conditions. However, other mRNA segments, for which exonucleolytic degradation was postulated to be rate-limiting, decayed with the same rate under either high or low oxygen tension. Likewise, the degradation of the puhA mRNA, the cycA mRNA, and the cytfbc mRNA was found to be independent of the oxygen tension in the culture. The data strongly suggest that specific mRNA sequences or structures are responsible for the observed oxygen effect on mRNA stability.
Blotting, Northern↗
A DNA sequence upstream of the puf operon of Rhodobacter capsulatus is involved in its oxygen-dependent regulation and functions as a protein binding site.
The transcription of the polycistronic puf operon which encodes pigment binding proteins of the reaction center and light-harvesting complex I of Rhodobacter capsulatus is regulated by the oxygen tension in the culture. A DNA sequence upstream of the puf transcriptional start was identified as a protein binding site. A DNA fragment carrying this DNA sequence participated in the formation of two DNA-protein complexes. The relative amounts of the two complexes were dependent on the oxygen tension in cultures from which the cytosolic fraction used for the in vitro binding studies was isolated. A single base pair transition within the protein binding site affected the oxygen-dependent expression of puf in vivo and the formation of DNA-protein complexes in vitro. The data suggest that the formation of specific DNA-protein complexes is involved in the oxygen-dependent regulation of the puf promoter. A DNA fragment containing the promoter region of the puc operon that encodes proteins of the light-harvesting complex II acted as a competitor for the formation of the DNA-protein complexes with the puf-specific fragment, indicating coregulation of the two operons.
Bacterial Proteins↗
Light and oxygen effects share a common regulatory DNA sequence in Rhodobacter capsulatus.
External factors regulate the formation of pigment protein complexes in facultatively photosynthetic bacteria. The puf operon of Rhodobacter capsulatus encodes the pigment binding proteins of the reaction centre and light-harvesting I complex. Here we demonstrate that a single base-pair exchange within a sequence of dyad symmetry upstream of the puf promoter affects both the oxygen regulation and the light regulation of the formation of reaction-centre and light-harvesting I complexes in Rhodobacter capsulatus. Our results imply that effects of oxygen or light ultimately act on the same regulatory DNA sequence, although it is still unknown how these environmental signals are sensed and transmitted to a transcriptional regulator.
Aerobiosis↗
A base pair transition in a DNA sequence with dyad symmetry upstream of the puf promoter affects transcription of the puc operon in Rhodobacter capsulatus.
A DNA sequence with dyad symmetry upstream of the transcriptional start of the Rhodobacter capsulatus puf operon, which encodes pigment-binding proteins of the light-harvesting I complex and of the reaction center, has previously been shown to be a protein-binding site (G. Klug, Mol. Gen. Genet. 226:167-176, 1991). When a low-copy-number plasmid with a base pair transition at position -43 within this dyad symmetry in front of the puf structural genes was transferred into a Rhodobacter strain with the puf operon deleted, different phenotypes occurred during cultivation of the transconjugants and the kinetics of the loss of the wild-type phenotype was dependent on the oxygen tension in the culture. After growth for 150 generations, the different phenotypes were stably inherited. The strains having the wild-type phenotype carried the wild-type puf DNA sequence. The original mutation was still present in the strains that showed lighter color. These strains had less light-harvesting II complex in the membrane and showed lower rates of transcription of the puc operon, which encodes the proteins of this complex. This deregulation of puc expression was due to one or more chromosomally located, secondary mutations, not directly to the mutation present on the plasmid. Thus, a single-base-pair transition in the puf upstream region can result in a deregulation of puc expression, suggesting a direct or indirect transcriptional coregulation of both these operons by a common factor.
Bacterial Proteins↗
Effects of translation on degradation of mRNA segments transcribed from the polycistronic puf operon of Rhodobacter capsulatus.
Previous work has shown that expression of genes within the polycistronic puf operon of Rhodobacter capsulatus is regulated in part by differential degradation of segments of puf transcripts. To understand the role of ribosome coverage in the differential stability of puf mRNA segments, we have studied the effects of mutations that alter translation of specific puf transcript segments on puf mRNA decay. Our results show that stopping translation either within the light-harvesting I (LHI) genes or near the 5' end of the reaction center (RC)-coding region decreased the stability of puf transcript segments downstream from a hairpin loop structure located between the LHI and RC genes but failed to affect the upstream sequences so long as the loop was present. Mutations that allowed translation to proceed through the hairpin structure reduced its ability to protect upstream sequences from accelerated decay. Introduction of translation stops more than 107 bp into the RC-coding region, but still 5' to an mRNA segment containing decay-promoting endonuclease cleavage sites, had no effect on puf mRNA stability. The divergent and location-dependent consequences of translation stops imply that different mechanisms are responsible for the degradation of different puf mRNA segments and indicate that coverage of puf mRNA sequences by ribosomes is insufficient and may in some cases be unnecessary to protect these sequences from degradation.
Blotting, Northern↗
Effects on the formation of antenna complex B870 of Rhodobacter capsulatus by exchange of charged amino acids in the N-terminal domain of the alpha and beta pigment-binding proteins.
The N-terminal domains of the alpha and beta polypeptides of the B870 antenna complex of Rhodobacter capsulatus are oppositely charged. In both polypeptides two charged amino acids are located close to the N-terminus, and two of them are close to the hydrophobic central domain. To test the hypothesis that charged amino acids in the N-terminus have a function for insertion and assembly of pigment-binding polypeptides, charged amino acids were replaced by amino acids of opposite charge. The results show that an exchange of amino acid positions 3 and 6 in alpha (Lys----Glu) or 2 and 5 in beta (Asp----Lys, Arg) has little effect under semiaerobic conditions on the formation of B870 but the additional exchange of positions 14 and 15 in alpha (Arg----Glu, Asp) and/or 13 and 14 in beta (Asp, Glu----Arg) inhibits strongly under semiaerobic dark and anaerobic light conditions the stable incorporation of the polypeptides into the membrane and the formation of the B870 complex. The mutant U43(pTXAB5) is able to grow without any antenna.
Amino Acid Sequence↗
A negatively charged N terminus in the alpha polypeptide inhibits formation of light-harvesting complex I in Rhodobacter capsulatus.
Light-harvesting complex I (LHI) of Rhodobacter capsulatus contains bacteriochlorophyll and carotenoids which are noncovalently bound to two different apoproteins (alpha and beta polypeptides) carrying oppositely charged N-terminal ends. The contribution of these charged segments to the assembly of LHI was studied with mutants having oppositely charged amino acids in the alpha or beta polypeptide. The influence of these mutations on the insertion and assembly process of the LHI complex was investigated by means of spectroscopic analysis of isolated intracytoplasmic membranes and pulse-chase experiments. Exchange of four positively charged amino acids to negatively charged amino acids on the N-terminal domain of the alpha subunit inhibited completely the assembly of the LHI complex. Although this mutant has no antenna, the reaction center is active and the cells were able to grow anaerobically in the light. Conversely, mutation of the four negatively charged amino acids of the N-terminal segment of the beta polypeptide did not prevent the assembly of the LHI complex, although the stability of the complex and the size of the photosynthetic unit were affected. The presence of the mutated beta polypeptide was confirmed by protein sequencing.
Amino Acid Sequence↗
Combined actions of multiple hairpin loop structures and sites of rate-limiting endonucleolytic cleavage determine differential degradation rates of individual segments within polycistronic puf operon mRNA.
Differential expression of the genes within the puf operon of Rhodobacter capsulatus is accomplished in part by differences in the rate of degradation of different segments of the puf transcript. We report here that decay of puf mRNA sequences specifying the light-harvesting I (LHI) and reaction center (RC) photosynthetic membrane peptides is initiated endoribonucleolytically within a discrete 1.4-kilobase segment of the RC-coding region. Deletion of this segment increased the half-life of the RC-coding region from 8 to 20 min while not affecting decay of LHI-coding sequences upstream from an intercistronic hairpin loop structure shown previously to impede 3'-to-5' degradation. Prolongation of RC segment half-life was dependent on the presence of other hairpin structures 3' to the RC region. Inserting the endonuclease-sensitive sites into the LHI-coding segment markedly accelerated its degradation. Our results suggest that differential degradation of the RC- and LHI-coding segments of puf mRNA is accomplished at least in part by the combined actions of RC region-specific endonuclease(s), one or more exonucleases, and several strategically located exonuclease-impeding hairpins.
Base Sequence↗
Pleiotropic effects of localized Rhodobacter capsulatus puf operon deletions on production of light-absorbing pigment-protein complexes.
The polycistronic puf operon of Rhodobacter capsulatus encodes protein components for the photosynthetic reaction center and one of the two antenna complexes involved in the capture of light energy. We report here that deletions within specific puf genes alter the synthesis and/or assembly in the photosynthetic membranes of pigment-protein complexes not affected genetically by the deletion. The pufX gene is required for normal ratios of antenna complexes, and its deletion results in an increase of membrane-bound light-harvesting I (LHI) complex-specific proteins. Expression of pufQ in strains deleted for the genes encoding the LHI and the photosynthetic reaction center (RC) yields a novel A868 peak that has not been associated with any of the pigment-protein complexes described previously. While deletions in the RC-coding region resulted in decreased LHI absorbance, no quantitative alteration in membrane-bound LHI protein was observed, suggesting that an intact RC complex is required for correct assembly of LHI in the membrane.
Bacterial Proteins↗
Biological consequences of segmental alterations in mRNA stability: effects of deletion of the intercistronic hairpin loop region of the Rhodobacter capsulatus puf operon.
It has been proposed that intercistronic stem and loop structures located in the puf operon of the photosynthetic bacterium Rhodobacter capsulatus account for segmental differences in transcript stability and consequently, the differential expression of the B870 and reaction center (RC) proteins encoded by puf. We report here that deletion of these structures leads to a failure to detect as discrete fragments the B870-encoding 0.49 kb and 0.50 kb mRNA segments located upstream from the site of the hairpins. The absence of these stable transcript fragments is associated with altered stoichiometry of the B870 and RC pigment-protein complexes in the bacterial intracytoplasmic membrane and a decreased rate of cell growth under photosynthetic conditions. These results support the view that the hairpin loop structures of the puf intercistronic region function in vivo to impede exoribonucleolytic degradation of upstream mRNA and establish that segmental variations in mRNA stability have a biologically important role in regulating the expression of puf operon genes.
Base Sequence↗
Microcompartmentation at the mitochondrial surface: its function in metabolic regulation.
Explore the source record for details and available documents.
Animals↗
Decreased parvalbumin contents in skeletal muscles of C57BL/6J(dy2J/dy2J) dystrophic mice.
Parvalbumin content is decreased by 40% in hindlimb muscles of adult (3.5 months) dystrophic mice when compared to normal mice of the same age. The concentration of parvalbumin in the dystrophic muscles resembles that of immature muscles of 4-week-old normal mice. The reduction may contribute to an elevation of sarcoplasmic free CA2+, which could stimulate various Ca2+-dependent processes related to the disease.
Animals↗
Alterations in phenotype expression of muscle by chronic nerve stimulation.
Explore the source record for details and available documents.
Animals↗
Parvalbumin reduction in relation to possible perturbations of Ca2+-homeostasis in muscular dystrophy.
Explore the source record for details and available documents.
Animals↗