Genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete Corynebacterium glutamicum

Abstract

The genome-wide identification of transcription start sites, enabled by high-throughput sequencing of a cDNA library enriched for native 5′ transcript ends, is ideally suited for the analysis of promoters. Here, the transcriptome of Corynebacterium glutamicum, a non-pathogenic soil bacterium from the actinomycetes branch that is used in industry for the production of amino acids, was analysed by transcriptome sequencing of the 5′-ends of native transcripts. Total RNA samples were harvested from the exponential phase of growth, therefore the study mainly addressed promoters recognized by the main house-keeping sigma factor σA. The identification of 2454 transcription start sites (TSS) allowed the detailed analysis of most promoters recognized by σA and furthermore enabled us to form different promoter groups according to their location relative to protein-coding regions. These groups included leaderless transcripts (546 promoters), short-leadered (<500 bases) transcripts (917), and long-leadered (>500 bases) transcripts (173) as well as intragenic (557) and antisense transcripts (261). All promoters and the individual groups were searched for information, e.g. conserved residues and promoter motifs, and general design features as well as group-specific preferences were identified. A purine was found highly favored as TSS, whereas the −1 position was dominated by pyrimidines. The spacer between TSS and ‐10 region were consistently 6–7 bases and the −10 promoter motif was generally visible, whereas a recognizable −35 region was only occurring in a smaller fraction of promoters (7.5%) and enriched for leadered and antisense transcripts but depleted for leaderless transcripts. Promoters showing an extended −10 region were especially frequent in case of non-canonical −10 motifs (45.5%). Two bases downstream of the −10 core region, a G was conserved, exceeding 40% abundance in most groups. This fraction reached 74.6% for a group of putative σB-dependent promoters, thus giving a hint to a specific property of these promoters. In addition, the high number of promoters analysed allowed finding of subtle signals only showing up significantly with this large set. This included the observation of a periodically changing A+T-content with maxima spaced by a full turn of the DNA helix. This periodic structure includes the A+T-rich UP-element of bacterial promoters known before but was found to extend up to −100, indicating hitherto unknown constraints influencing promoter architecture and possibly also promoter function.