Global analysis of the RpaB regulon based on the positional distribution of HLR1 sequences and comparative differential RNA-Seq data

Abstract

21 expression, regulatory networks, sRNAs 22 23 The transcription factor RpaB regulates the expression of genes encoding 24 photosynthesis-associated proteins during light acclimation. The binding site of RpaB 25 is the HLR1 motif, a pair of imperfect octameric direct repeats, separated by two 26 random nucleotides. Here, we used high-resolution mapping data of transcriptional 27 start sites (TSSs) in the model Synechocystis sp. PCC 6803 in conjunction with the 28 positional distribution of HLR1 sites for the global prediction of the RpaB regulon. The 29 results demonstrate that RpaB regulates the expression of more than 150 promoters, 30 driving the transcription of protein-coding and non-coding genes and antisense 31 transcripts under low light and upon the shift to high light when DNA binding activity is 32lost. Transcriptional activation by RpaB is achieved when the HLR1 motif is located 66 33 to 45 nt upstream, repression occurs when it is close to or overlapping the TSS. 34 Selected examples were validated by multiple experimental approaches, including 35 chromatin affinity purification, reporter gene, northern hybridization and electrophoretic 36 mobility shift assays. We found that RpaB controls ssr2016/pgr5, which is involved in 37 cyclic electron flow and state transitions; six out of nine ferredoxins; three of four FtsH 38 proteases; gcvP/slr0293, encoding a crucial photorespiratory protein; and nirA and isiA 39 for which we suggest cross-regulation with the transcription factors NtcA or FurA, 40 respectively. In addition to photosynthetic gene functions, RpaB contributes to the 41 control of genes affiliated with nitrogen assimilation, cofactor biosyntheses, the 42 CRISPR system and the circadian clock, making it one of the most versatile regulators 43 in cyanobacteria.44 Significance Statement: RpaB is a transcription factor in cyanobacteria and in the 45 chloroplasts of several lineages of eukaryotic algae. Like other important transcription 46 factors, the gene encoding RpaB cannot be deleted, making the study of deletion 47 54 RpaB ("regulator of phycobilisome association B") is an OmpR-type transcription factor 55 of crucial importance for the transcriptional control of multiple photosynthesis-56 associated genes under varying light conditions. RpaB was discovered in 57 Synechocystis sp. PCC 6803 (from here: Synechocystis 6803) based on its ability to 58 affect the energy distribution from phycobilisomes (PBS) to photosystem I (PSI) 59 relative to photosystem II (PSII) (1). Orthologs of rpaB belong to the cyanobacterial 60 core genome and were discovered in the chloroplast genomes of all non-green algae 61 except Odontella (2) and in the charophyte alga Chlorokybus atmophyticus (see (3) for 62 a recent review), suggesting important and evolutionarily widely conserved functions.63The rpaB gene cannot be deleted by conventional methods, indicating its essentiality 64 (1, 4-7). Despite its wide distribution, most insight into the genes controlled by RpaB 65 have been obtained from analyses ...