Kulbachinskiy, A. | Search bioRxiv

bioRxiv2018

The expanded universe of prokaryotic Argonaute proteins

The members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition - and sometimes cleavage - of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and almost tripled the number of previously analyzed genes of this family. In comparison with eAgos, pAgos are highly diverse and have likely spread by horizontal gene transfer. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids and in target cleavage, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and processing. Many pAgos, including catalytically inactive variants, are associated with putative nucleases, helicases and DNA binding proteins in the same gene or operon, suggesting that they are involved in DNA processing. The great diversity of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and their use as potential tools in genome editing.

genomics↗

bioRxiv2017

Pausing controls branching between productive and non-productive pathways during initial transcription

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region {sigma}3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

biophysics↗