Rhodobacter Capsulatus Puf Operon Research Articles

Effect of the pufQ-pufB intercistronic region on puf mRNA stability in Rhodobacter capsulatus.

Differential expression of genes localized within the polycistronic puf operon of Rhodobacter capsulatus is partly due to altered stabilities of individual mRNA segments. We show that the 5' untranslated region (UTR) of pufB contributes to the unusual longevity of the 0.5 kb light-harvesting (LH) I specific pufBA mRNA and of the 2.7 kb pufBALMX mRNA. Three stem-loop structures have been identified within the pufQ-pufB intercistronic region by means of RNA secondary-structure analysis in vitro and in vivo. Deletion analysis of the pufB 5' UTR indicates that the complete set of secondary structures is required to maintain wild-type levels of pufBA mRNA stability. A phylogenetic comparison of pufB 5' UTRs of other photosynthetic bacteria reveals an evolutionary conservation of the base-pairing potential despite sequence divergence. Comparison of puf mRNA decay in Escherichia coli strains with or without endoribonuclease E (RNase E) activity suggests that the pufB 5' secondary structures protect the downstream mRNA segment against degradation by RNase E. Removal of the 117-nucleotide pufQ-pufB intercistronic region results in loss of stability for the pufBA and pufBALMX mRNAs with concomitant stabilization of the full-length puf primary transcript (QBALMX). We therefore conclude that the deleted sequence functions both as a stabilizing element for pufBALMX and pufBA segments and as a target site for initial rate-limiting decay of the unstable pufQBALMX mRNA.

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3' of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3' end of pufM and sequences past the end of pufM.

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.

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.

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.

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.

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.

Isolation and characterization of Rhodobacter capsulatus mutants defective in oxygen regulation of the puf operon

cis-acting mutations that affect regulation of the Rhodobacter capsulatus puf operon by oxygen were isolated by placing the mutagenized puf regulatory region 5' to a promoterless Tn5 neo gene, which encodes resistance to kanamycin (Kmr). R. capsulatus mutants that failed to show wild-type repression of KMr by oxygen were selected and analyzed. Four independent clones contained point mutations, three of which were identical, in a region of dyad symmetry located between puf operon nucleotide positions 177 and 207, approximately 45 base pairs 5' to the site of initiation of puf transcripts. The phenotypic effects of the aerobically selected mutations were duplicated by single and double point mutations introduced site specifically into the region of dyad symmetry by oligonucleotide-directed mutagenesis. Determinations of the bacterial 50% lethal dose of kanamycin, of aminoglycoside phosphotransferase activity in cell sonicates, and of neo-specific mRNA confirmed the diminished responsiveness of the mutants to oxygen and consequently implicated the mutated region in O2-mediated transcriptional regulation.

Structural and functional analysis of transcriptional control of the Rhodobacter capsulatus puf operon

We report data indicating that the Rhodobacter capsulatus puf operon promoter and the site for its oxygen regulation are located more than 700 base pairs upstream from the previously identified puf genes and have identified the nucleotide sequences that constitute these control signals. A model is proposed in which a polycistronic transcript at least 3.4 kilobases in length is initiated near the O2-regulated promoter and is processed posttranscriptionally by endonucleolytic cleavage at multiple sites, yielding discrete mRNA segments that are degraded at different rates. A newly identified gene (pufQ), which includes a hydrophobic domain having some similarity to domains of the products of the pufL and pufM genes, begins 313 nucleotides into the puf transcript and is located entirely within the most rapidly degraded segment of the transcript. A previously identified puf transcript segment encoding structural proteins for photosynthetic membrane complexes persists after degradation of the most 5' region of the transcript and is itself subject to segmentally specific degradation. Our results suggest a model in which differential expression of the multiple genes encoded by the puf operon is at least in part attributable to major differences in the rates of decay of the various segments of puf mRNA.

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.

Analysis of the Rhodobacter capsulatus puf operon. Location of the oxygen-regulated promoter region and the identification of an additional puf-encoded gene.

In an attempt to identify features of an oxygen-regulated promoter, we have determined the location of transcription initiation for the puf operon. The position for the oxygen-regulated promoter was demonstrated by several independent means to be located 699 base pairs (bp) upstream from the pufB structural gene. DNA sequence analysis of the promoter region demonstrates the presence of a 26-base pair region of dyad symmetry followed by a sequence containing homology to promoters which use the RNA polymerase sigma 60 subunit (ntrA) for recognition of DNA. In addition to the oxygen-regulated promoter, a region responsible for low-level constitutive expression of the puf operon was shown to initiate transcription 511 bp upstream from the pufB gene. In contrast to the oxygen-regulated promoter, this second promoter contains no obvious secondary structure nor sequence homology to ntrA-dependent promoters. DNA sequence analysis demonstrates the existence of an additional open reading frame (designated as pufQ) that is located between the promoters and the pufB structural gene. A translational fusion of pufQ to lacZ was used to demonstrate that pufQ is efficiently translated and regulated in a manner analogous to a translational fusion of pufM to lacZ. Finally, we also demonstrate that puf operon transcription initiation and regulation does not involve any puf-encoded gene products.

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.