Transcription termination sets the 3’ end boundaries of RNAs and plays key roles in gene regulation. While termination has been well studied in model bacteria, the signals that mediate termination in archaea, the third domain of life, remain poorly understood. Here we applied term-seq to comprehensively map RNA 3’ termini, with single base precision, in two phylogenetically distant archaea: Methanosarcina mazei and Sulfolobus acidocaldarius. Comparison of RNA 3’ ends across hundreds of genes revealed the sequence composition of typical transcriptional terminators in each organism, highlighting both common and divergent characteristics between the different archaeal phyla. We find that in contrast to bacteria, a considerable portion of archaeal genes are controlled by multiple consecutive terminators, generating several alternative 3’UTRs isoforms for over 30% of the genes. These alternative isoforms often present marked length differences, implying that archaea can employ regulation via alternative 3’UTRs similar to eukaryotes. While most of the terminators are intergenic, we discover numerous cases in which termination of one gene occurs within the coding region of a downstream gene, implying that leaky termination may tune transcript stoichiometry of individual genes organized in multi-gene operons regulated by a single promoter. These results provide the first high-throughput maps of transcriptional terminators in archaea, and point to an evolutionary path that links between the bacterial and eukaryal non-coding regulatory strategies.
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