0000000000277002
AUTHOR
Maria L. Salvador
Structural and functional characterization of a transcription-enhancing sequence element in the rbcL gene of the Chlamydomonas chloroplast genome.
The structure and function of a transcription-enhancing sequence element in the coding region of the Chlamydomonas reinhardtii rbcL gene was analyzed in Chlamydomonas chloroplast transformants in vivo. The enhancer sequence is contained within a DNA segment extending from position +108 to position +143, relative to the start site of rbcL gene transcription. The sequence remains functional when inverted or when placed 34 bp closer to or 87 bp further downstream of the basic rbcL promoter. However, it does not function from a site about 250 bp downstream of its original location. Besides promoting transcription initiation from the rbcL promoter, the element is able to augment transcription fr…
Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii
Using a series of point mutations in chimeric reporter gene constructs consisting of the 5' regions of the Chlamydomonas chloroplast rbcL or atpB genes fused 5' to the coding sequence of the bacterial uidA (GUS) gene, RNA-stabilizing sequence elements were identified in vivo in the 5' untranslated regions (5' UTRs) of transcripts of the chloroplast genes rbcL and atpB in Chlamydomonas reinhardtii. In chimeric rbcL 5' UTR:GUS transcripts, replacement of single nucleotides in the 10-nt sequence 5'-AUUUCCGGAC-3', extending from positions +38 to +47 relative to the transcripts' 5' terminus, shortened transcript longevity and led to a reduction in transcript abundance of more than 95%. A similar…
Processing of RNAs of the Family Avsunviroidae in Chlamydomonas reinhardtii Chloroplasts
ABSTRACT The family Avsunviroidae comprises four viroid species with the ability to form hammerhead ribozymes that mediate self-cleavage of the multimeric plus and minus strands resulting from replication in the chloroplast through a symmetric rolling-circle mechanism. Research on these RNAs is restricted by their host range, which is limited to the plants wherein they were initially identified and some closely related species. Here we report cleavage and ligation in transplastomic Chlamydomonas reinhardtii expressing plus- and minus-strand dimeric transcripts of representative members of the family Avsunviroidae . Despite the absence of viroid RNA-RNA transcription, the C. reinhardtii -bas…
Specific roles of 5′ RNA secondary structures in stabilizing transcripts in chloroplasts
RNA secondary structures, e.g. stem-loops that are often found at the 5' and 3' ends of mRNAs, are in many cases known to be crucial for transcript stability but their role in prolonging the lifetime of transcripts remains elusive. In this study we show for an essential RNA-stabilizing stem-loop at the 5' end of rbcL gene transcripts in Chlamydomonas that it neither prevents ribonucleases from binding to the RNA nor impedes their movement along the RNA strand. The stem-loop has a formative function in that it mediates folding of a short sequence around its base into a specific RNA conformation, consisting of a helical and single-stranded region, i.e. the real structure required for longevit…
The redox state regulates RNA degradation in the chloroplast of Chlamydomonas reinhardtii.
Abstract A Chlamydomonas reinhardtii chloroplast transformant, designated MU7, carrying a chimeric (rbcL promoter: β-glucuronidase [GUS]:psaB 3′ end) gene whose transcripts have been found previously to be unstable in light (half-life of 20 min in light as opposed to a half-life of 5 h in the dark), was used to study the role of electron transport and of the redox state in the degradation of chloroplast transcripts in the light. Blocking photosynthetic electron transport with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented the light-dependent breakdown of the pool of GUS transcripts in MU7 cells. Diamide, an oxidizing agent, caused a measurable delay in the degradation of GUS trans…
Interactive effects between physical forces and ecoystem engieneers on seed burial: a case study using Spartina anglica
Seed burial (i.e. vertical seed dispersal) has become increasingly valued for its relevance for seed fate and plant recruitment. While ecosystem engineers have been generally considered as the most important drivers of seed burial, the role of physical forces, such as wind or water flow, has been largely overlooked. Using tidal habitats as a model system, and a combination of flume and mesocosm experiments, we investigated the effects of 1) currents, 2) benthic animals with different engineering activities and 3) their interplay on seed burial of a common salt marsh pioneer plant, Spartina anglica. Our results reveal that in such systems, water flow can be of equal or higher importance than…
Endogenous fluctuations of DNA topology in the chloroplast of Chlamydomonas reinhardtii.
DNA supercoiling in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii was found to change with a diurnal rhythm in cells growing in alternating 12-h dark-12-h light periods. Highest and lowest DNA superhelicities occurred at the beginning and towards the end of the 12-h light periods, respectively. The fluctuations in DNA supercoiling occurred concurrently and in the same direction in two separate parts of the chloroplast genome, one containing the genes psaB, rbcL, and atpA and the other containing the atpB gene. Fluctuations were not confined to transcribed DNA regions, indicating simultaneous changes in DNA conformation all over the chloroplast genome. Because the d…
Changes in the 5?-untranslated region of the rbcL gene accelerate transcript degradation more than 50-fold in the chloroplast of Chlamydomonas reinhardtii
Using uidA (beta-glucuronidase; GUS) reporter gene constructs, the 5'-untranslated region (UTR) of the Chlamydomonas chloroplast rbcL gene was screened by deletion and mutational analysis for the presence of a promoter element that previous studies implied to reside within the first 63 base pairs of the UTR. Deleting a large segment of the rbcL 5'UTR in a 3'--5' direction to position +36, changing the remaining 36 base pairs at the 5' end of the UTR, and increasing by five base pairs the distance between the rbcL 5'UTR and the basic promoter element located at position -10 did not abolish transcription from the basic rbcL promoter. It is concluded that the apparent loss of transcriptional a…
Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast.
Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was …
Messenger RNA degradation is initiated at the 5′ end and follows sequence- and condition-dependent modes in chloroplasts
Using reporter gene constructs, consisting of the bacterial uidA (GUS) coding region flanked by the 5' and 3' regions of the Chlamydomonas rbcL and psaB genes, respectively, we studied the degradation of mRNAs in the chloroplast of Chlamydomonas reinhardtii in vivo. Extending the 5' terminus of transcripts of the reporter gene by more than 6 nucleotides triggered rapid degradation. Placing a poly(G) tract, known to pause exoribonucleases, in various positions downstream of the 5' terminus blocked rapid degradation of the transcripts. In all these cases the 5' ends of the accumulating GUS transcripts were found to be trimmed to the 5' end of the poly(G) tracts indicating that a 5' → 3' exori…