Spatial And Temporal Organization Of Nucleoid,RNA Polymerase And Ribosome In The Cyanobacterium Synechococcus Elongatus PCC 7942

Duplication,transcription and translation are the most fundamental processes for the life.In eukaryotic cells,transcriptional and translational activity is largely regulated by chromosomal packaging.In prokaryotes,although there is no nuclear envelope structure and DNA-packaging proteins,the chromosome is highly condensed into nucleoid.At present,the spatial organization among nucleoid,transcription and translation and the effect of them on DNA organization remain poorly understood.The circadian rhythm is an endogenous time mechanism whose function is to regulate cell population metabolism and various behavioral activities during the circadian cycle.Synechococcus sp.PCC 7942 is the main model organism for studying the molecular mechanism and regulation of the circadian rhythm.Based on the physiology of cyanobacteria,the research explored the spatial organization of nucleoid,RNA polymerase and ribosome in Synechococcus sp.PCC 7942 by means of fluorescent protein tags.The main research content and results are as follows:Firstly,the RpoC2 encode the ?' subunit of RNAP,and RNAPs can be tagged by insert the eYFP fluorescent protein and gentamicin antibiotic coding sequence on the downstream of RpoC2.Ribosome-labeling stain was constructed in the same way as the RNAP-labeling stain as previously described.We tracked the spatial arrangement of DNA/RNAPs/ribosomes within 96 hours and found that DNA/RNAPs/ribosomes had a rhythmic arrangement with a 24-hour cycle.The intracellular spatio-temporal distribution of RNAP is synchronous with the expansion and compression of the nucleoid.Spatially,the fluorescent labeling signal is slightly more extended than the DAPI-stained signal.Ribosomes form a highly complementary spatial pattern with nucleoids during DNA compression.Secondly,through inhibition of the translational inhibitor chloramphenicol and transcription inhibitor rifampicin,fluorescence signal tracking revealed that in the case of inhibition of the translation process,intracellular DNA was highly compressed,the ribosome and DNA were separated in space,and concentrated in the thylakoid membrane region.After the transcription process was suppressed,the space of RNA polymerase is always similar to DNA arrangement,and the ribosome signal(small subunits)eventually flooded the entire cell.Combined with the rich membrane structure of cyanobacteria,our experiments provide more intuitive evidence for the transertion hypothesis.Thirdly,the variation trend of intracellular total content of RNA polymerase and ribosome in Synechococcus sp.PCC 7942 were measured by flow cytometry.The results indicate that RNAP copy number is generally stable during growth and exhibits a rhythm that fluctuates around a fixed value.However,ribosome copy number exhibits intensive increase and moderate reduction.