Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases

RNA cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in bacterial stress adaptation.</p

Catalytically active Argonaute nuclease from Synechococcus elongatus

Argonaute proteins, which are found in almost all eukaryotes and in many prokaryotes, use small nucleic acid guides for the recognition and cleavage of complementary nucleic acids. While the role of eukaryotic Argonautes in RNA interference is well understood, the functions of prokaryotic Argonautes remain largely unknown. It was proposed that they may provide defense against invading nucleic acids, preferably acting on DNA targets. In this work, we studied the SynAgo protein from the cyanobacterium Synechococcus elongatus. We expressed affinity-tagged SynAgo in S. elongatus, purified the protein, and sequenced and mapped associated nucleic acids. We showed that SynAgo is bound with ~18 nt small DNAs coming from all genomic regions with no obvious gene specificity. Mass-spectrometry of copurified proteins from S. elongatus also revealed several possible protein partners of SynAgo. Biochemical analysis demonstrated that SynAgo is an active nuclease that can cleave both target DNA and RNA with varying efficiency, depending on the reaction conditions and the presence of mismatches between the guide and target strands. Finally, we introduced the SynAgo gene in the E. coli genome and tested its effects on plasmid maintenance and phage infections

Catalytically active Argonaute nuclease from Synechococcus elongatus

Argonaute proteins, which are found in almost all eukaryotes and in many prokaryotes, use small nucleic acid guides for the recognition and cleavage of complementary nucleic acids. While the role of eukaryotic Argonautes in RNA interference is well understood, the functions of prokaryotic Argonautes remain largely unknown. It was proposed that they may provide defense against invading nucleic acids, preferably acting on DNA targets. In this work, we studied the SynAgo protein from the cyanobacterium Synechococcus elongatus. We expressed affinity-tagged SynAgo in S. elongatus, purified the protein, and sequenced and mapped associated nucleic acids. We showed that SynAgo is bound with ~18 nt small DNAs coming from all genomic regions with no obvious gene specificity. Mass-spectrometry of copurified proteins from S. elongatus also revealed several possible protein partners of SynAgo. Biochemical analysis demonstrated that SynAgo is an active nuclease that can cleave both target DNA and RNA with varying efficiency, depending on the reaction conditions and the presence of mismatches between the guide and target strands. Finally, we introduced the SynAgo gene in the E. coli genome and tested its effects on plasmid maintenance and phage infections

AVALIAÇÃO EXPERIMENTAL DO PROTOCOLO 802.1X PARA PROVER MOBILIDADE E SEGURANÇA EM REDES DE COMPUTADORES

TCC (graduação) - Universidade Federal de Santa Catarina. Centro Tecnológico. Curso de Ciências da Computação.Este trabalho tem como proposta realizar um estudo sobre o protocolo 802.1x, observando seu funcionamento e características. Com base nesta pesquisa será realizada uma avaliação experimental deste protocolo, visando prover mobilidade e aumentar a segurança em rede de computadores

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small non-coding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small non-coding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes