Biomedical subjects

Jörg Nickelsen

Publications and source records attributed to Jörg Nickelsen.

8 recordsLinked to original sources

PubMed2006

Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon.

Translation initiation represents a key step during regulation of gene expression in chloroplasts. Here, we report on the identification and characterization of three suppressor point mutations which overcome a translational defect caused by the deletion of a U-rich element in the 5'-untranslated region (5'-UTR) of the psbD mRNA in the green alga Chlamydomonas reinhardtii. All three suppressors affect a secondary RNA structure encompassing the psbD AUG initiation codon within a double-stranded region as judged by the analysis of site-directed chloroplast mutants as well as in vitro RNA mapping experiments using RNase H. In conclusion, the data suggest that these new element serves as a negative regulator which mediates a rapid shut-down of D2 synthesis.

5' Untranslated Regions↗

PubMed2005

NAB1 is an RNA binding protein involved in the light-regulated differential expression of the light-harvesting antenna of Chlamydomonas reinhardtii.

Photosynthetic organisms respond to changes in ambient light by modulating the size and composition of their light-harvesting complexes, which in the case of the green alga Chlamydomonas reinhardtii consists of >15 members of a large extended family of chlorophyll binding subunits. How their expression is coordinated is unclear. Here, we describe the analysis of an insertion mutant, state transitions mutant3 (stm3), which we show has increased levels of LHCBM subunits associated with the light-harvesting antenna of photosystem II. The mutated nuclear gene in stm3 encodes the RNA binding protein NAB1 (for putative nucleic acid binding protein). In vitro and in vivo RNA binding and protein expression studies have confirmed that NAB1 differentially binds to LHCBM mRNA in a subpolysomal high molecular weight RNA-protein complex. Binding of NAB1 stabilizes LHCBM mRNA at the preinitiation level via sequestration and thereby represses translation. The specificity and affinity of binding are determined by an RNA sequence motif similar to that used by the Xenopus laevis translation repressor FRGY2, which is conserved to varying degrees in the LHCBM gene family. We conclude from our results that NAB1 plays an important role in controlling the expression of the light-harvesting antenna of photosystem II at the posttranscriptional level. The similarity of NAB1 and FRGY2 of Xenopus implies the existence of similar RNA-masking systems in animals and plants.

Amino Acid Sequence↗

PubMed2005

Relationship between mRNA levels and protein accumulation in a chloroplast promoter-mutant of Chlamydomonas reinhardtii.

The photosynthetic chloroplast mutant G64 of Chlamydomonas reinhardtii was shown to contain a single point mutation within the 5' region of the psbD gene encoding the D2 protein of the photosystem II reaction center. The mutation affects the sequence element TATAATAT which has previously been hypothesized to function as the psbD promoter. Run-on analysis confirmed that transcription of psbD in the mutant was reduced to approximately 10% of the wild-type level. However, psbD mRNA accumulated to approximately 35%, despite the prominent decrease in RNA synthesis. This suggests that RNA-stabilization effects can compensate to some extent for a reduction in transcriptional activity. Interestingly, a direct correlation between transcript levels and the accumulation of the psbD gene product, the D2-protein, was observed in G64. The data suggest that posttranscriptionally acting regulatory factors determine the rate-limiting steps of chloroplast psbD gene expression.

Amino Acid Sequence↗

PubMed2005

The yeast split-ubiquitin system to study chloroplast membrane protein interactions.

Each photosynthetic complex within the thylakoid membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein-protein interactions of thylakoid membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the thylakoid membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF(0)III, suggesting an important role of Alb3 in the assembly of photosynthetic thylakoid membrane complexes.

Arabidopsis↗

PubMed2005

Two adjacent nuclear genes are required for functional complementation of a chloroplast trans-splicing mutant from Chlamydomonas reinhardtii.

The chloroplast tscA gene from Chlamydomonas reinhardtii encodes a co-factor RNA that is involved in trans-splicing of exons 1 and 2 of the psaA mRNA encoding a core polypeptide of photosystem I. Here we provide molecular and genetic characterization of the trans-splicing mutant TR72, which is defective in the 3'-end processing of the tscA RNA and consequently defective in splicing exons 1 and 2 of the psaA mRNA. Using genomic complementation, two adjacent nuclear genes were identified, Rat1 and Rat2, that are able to restore the photosynthetic growth of mutant TR72. Restoration of the photosynthesis phenotype, however, was successful only with a DNA fragment containing both genes, while separate use of the two genes did not rescue the wild-type phenotype. This was further confirmed by using a set of 10 gene derivatives in complementation tests. The deduced amino acid sequence of Rat1 shows significant sequence homology to the conserved NAD+-binding domain of poly(ADP-ribose) polymerases of eukaryotic organisms. However, mutagenesis of conserved residues in this putative NAD+-binding domain did not reveal any effect on restoration efficiency. Immunodetection analyses with enriched fractions of chloroplast proteins indicated that Rat1 is associated with chloroplast membranes. Using the yeast three-hybrid system, we were able to demonstrate the specific binding of tscA RNA by the Rat1 polypeptide. We propose that the two nuclear factors Rat1 and Rat2 are involved in processing of chloroplast tscA RNA and in subsequent splicing of psaA exons 1 and 2.

Algal Proteins↗

PubMed2004

PratA, a periplasmic tetratricopeptide repeat protein involved in biogenesis of photosystem II in Synechocystis sp. PCC 6803.

The light reactions of oxygenic photosynthesis are mediated by multisubunit pigment-protein complexes situated within the specialized thylakoid membrane system. The biogenesis of these complexes is regulated by transacting factors that affect the expression of the respective subunit genes and/or the assembly of their products. Here we report on the analysis of the PratA gene from the cyanobacterium Synechocystis sp. PCC 6803 that encodes a periplasmic tetratricopeptide repeat protein of formerly unknown function. Targeted inactivation of PratA resulted in drastically reduced photosystem II (PSII) content. Protein pulse labeling experiments of PSII subunits indicated that the C-terminal processing of the precursor of the reaction center protein D1 is compromised in the pratA mutant. Moreover, a direct interaction of PratA and precursor D1 was demonstrated by applying yeast two-hybrid analyses. This suggests that PratA represents a factor facilitating D1 maturation via the endoprotease CtpA. The periplasmic localization of PratA supports a model that predicts the initial steps of PSII biogenesis to occur at the plasma membrane of cyanobacterial cells.

Amino Acid Motifs↗

PubMed2003

Chloroplast RNA-binding proteins.

Chloroplast gene expression is regulated by nucleus-encoded factors, which mainly act at the post-transcriptional level. Plastid RNA-binding proteins (RBPs) represent good candidates for mediating these functions. The picture emerging from recent analyses is that of a great number of differentially regulated RBPs, which are organized in distinct, spatially separated supramolecular complexes. This reflects the complexity of the regulatory network that underlies the intracellular communication system between the nucleus and the chloroplast.

5' Untranslated Regions↗

PubMed2002

A chloroplast RNA binding protein from stromal thylakoid membranes specifically binds to the 5' untranslated region of the psbA mRNA.

The intrachloroplastic localization of post-transcriptional gene expression steps represents one key determinant for the regulation of chloroplast development. We have characterized an RNA binding protein of 63 kDa (RBP63) from Chlamydomonas reinhardtii chloroplasts, which cofractionates with stromal thylakoid membranes. Solubility properties suggest that RBP63 is a peripheral membrane protein. Among RNA probes from different 5' untranslated regions of chloroplast transcripts, RBP63 preferentially binds to the psbA leader. This binding is dependent on a region comprising seven consecutive A residues, which is required for D1 protein synthesis. A possible role for this newly discovered RNA binding protein in membrane targeting of psbA gene expression is discussed.

5' Untranslated Regions↗