Uwe Klein

Endogenous Fluctuations of DNA Topology in the Chloroplast of Chlamydomonas reinhardtii

ABSTRACT DNA supercoiling in the chloroplast of the unicellular green algaChlamydomonas 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, andatpA 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 diurnal fluctuations persisted in cells kept in continuous light, DNA supercoiling is judged to be under endogenous control. The endogenous fluctuations in chloroplast DNA topology correlated tightly with the endogenous fluctuations of overall chloroplast gene transcription and with those of the pool sizes of most chloroplast transcripts analyzed. This result suggests that DNA superhelical changes have a role in the regulation of chloroplast gene expression in Chlamydomonas.

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 longevity of rbcL transcripts in chloroplasts. Disturbing this structure renders transcripts completely unstable, even if the sequence of this element is not altered. The requirement of a specific 5′ sequence and structure for RNA longevity suggests an interaction of this element with a trans-acting factor that protects transcripts from rapid degradation in chloroplasts.

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′ exoribonuclease is involved in the degradation process. Several unstable variants of the GUS transcript could not be rescued from rapid degradation by a poly(G) tract showing that sequence/structure-dependent modes of mRNA breakdown exist in the Chlamydomonas chloroplast. Furthermore, degradation of poly(G)-stabilized transcripts that accumulated in cells maintained in the dark could be augmented by illuminating the cells, implying a photo-activated mode of mRNA degradation that is not blocked by a poly(G) tract. These results suggest sequence- and condition-dependent 5′→3′ mRNA-degrading pathways in the chloroplast of C. reinhardtii.

Chapter 25 – Chloroplast Transcription

Publisher Summary This chapter envisages the single gene and gene cluster transcription units. To date, three types of chloroplast promoters have been identified in Chlamydomonas by deletion, mutational, and functional analyses. The Chlamydomonas chloroplast genome contains about 100 genes that are organized and transcribed either singly or as part of multi-gene transcription units. In vitro and in vivo studies have identified sequence elements and proteins involved in transcription of several of these genes. The transcription machinery in Chlamydomonas shares many features with that of flowering plant chloroplasts, but also has a number of notable differences. Many Chlamydomonas chloroplast genes were found to be transcribed into monocistronic transcripts, but complex patterns of transcript accumulation do not seem to be uncommon. Transcription machineries are dealt in depth under chloroplast promoters, RNA polymerases with subunits and sigma subunits. Examples for both promoter sequence-dependent and regulative control of transcription initiation found in Chlamydomonas are described. Transcription is terminated when RNA polymerase pauses and the newly synthesized transcript is released from the ternary RNA polymerase/DNA/RNA complex. Transcription termination is overviewed and focus is also on the regulation of transcription. Understanding transcription and associated molecular processes might contribute to controlled expression of foreign genes in chloroplasts, and to informed and thoughtful manipulations of the chloroplast genome in biotechnological endeavors.