Role of RNA Secondary Structure and Processing in Stability of the nifH1 Transcript in the Cyanobacterium Anabaena variabilis

In the cyanobacterium Anabaena variabilis ATCC 29413 aerobic nitrogen fixation occurs in 17 microxic cells called heterocysts. Synthesis of nitrogenase in heterocysts requires expression of 18 the large nif1 gene cluster, which is primarily under the control of the promoter for the first 19 gene, nifB1. Strong expression of nifH1 requires the nifB1 promoter but is also controlled by 20 RNA processing, which leads to increased nifH1 transcript stability. The processing of the 21 primary nifH1 transcript occurs at the base of a predicted stem-loop structure that is conserved 22 in many heterocystous cyanobacteria. Mutations that changed the predicted secondary 23 structure or changed the sequence of the stem-loop had detrimental effects on the amount of 24 nifH1 transcript, with mutations that altered or destabilized the structure having the strongest 25 effect. Just upstream from the transcriptional processing site for nifH1 was the promoter for a 26 small antisense RNA, sava4870.1. This RNA was more strongly expressed in cells grown in the 27 presence of fixed nitrogen and was down regulated in cells 24 h after nitrogen step-down. A 28 mutant strain lacking the promoter for sava4870.1 showed delayed nitrogen fixation; however, 29 that phenotype might have resulted from an effect of the mutation on the processing of the 30 nifH1 transcript. The nifH1 transcript was the most abundant and most stable nif1 transcript, 31 while nifD1 and nifK1, just downstream of nifH1 were present in much lower amounts and were 32 less stable. The nifD1 and nifK1 transcripts were also processed at sites just upstream of nifD1 33 and nifK1.