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. 2010 Jul 25;38(21):7485–7499. doi: 10.1093/nar/gkq652

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© The Author(s) 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 10. — Premature transcription termination in the nitrogen-fixation genes. (A) Alignment of transcription termination-like signals in the nitrogen-fixation genes. It is likely that the nifEN operon contains nifEN and nifX in this order, the Gsul_0938 homologue operon contains a gene encoding a hypothetical protein, glnK and amtB in this order, and the Gsul_2799 homologue operon contains genes encoding a putative radical SAM domain protein and a putative acetyltransferase. The inverted-repeat sequences are underlined. Highly conserved nucleotides are indicated in bold letters. (B) Promoter region of nifH. Predicted GnfM-binding sites (Supplementary Table S4) are highlighted in gray. Highly conserved dinucleotides GG and GC in RpoN-dependent −24/−12 promoter elements are underlined. Transcription initiation site is indicated by +1. Transcription termination-like signals are indicated by arrows. Translation initiation codon is indicated by Met. (C) Putative RNA secondary structure of nifH. The putative stem-loop structure was predicted on the basis of base-pairing. A predicted binding site for GnfR is indicated in bold letters. (D) lacZ fusion assay. The assays were conducted with artificial promoters. Diagrams of promoter constructs are shown (see Supplementary Figure S5A for sequences). RpoD, the RpoD-dependent like promoter; RpoD-TTS (WT), the RpoD-dependent like promoter with the wild-type transcription termination signals from nifH; RpoD-TTS (-up), the RpoD-dependent like promoter with the transcription termination signals lacking the upstream half of the stem structure; RpoD-TTS (-down), the RpoD-dependent like promoter with the transcription termination signals lacking the downstream half of the stem structure; RpoD-TTS (mut), the RpoD-dependent like promoter with the transcription termination signals mutated in the stem structure (Supplementary Figure S5A). Mutations in RpoD-TTS (mut) were made by changing GGGGCGCCTT to ACATACAACA. β-Galactosidase activity was examined with the X-gal plate (Supplementary Figure S5B).