Harald Putzer

Bio: Harald Putzer is an academic researcher from University of Paris. The author has contributed to research in topics: RNase P & Bacillus subtilis. The author has an hindex of 28, co-authored 48 publications receiving 2545 citations. Previous affiliations of Harald Putzer include Paris Diderot University & Centre national de la recherche scientifique.

Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

Abstract: Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5' phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-beta-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation.

RNase Y, a novel endoribonuclease, initiates riboswitch turnover in Bacillus subtilis

Abstract: In contrast to Escherichia coli, initiation of mRNA decay in Gram-positive organisms is poorly understood. We studied the fate of the highly structured RNAs generated by premature transcription termination of S-adenosylmethionine (SAM)-dependent riboswitches in Bacillus subtilis. An essential protein of earlier unknown function, YmdA, was identified as a novel endoribonuclease (now called RNase Y) that was capable of preferential cleaving in vitro of the 5′ monophosphorylated yitJ riboswitch upstream of the SAM-binding aptamer domain. Antiterminated full-length yitJ mRNA was not a substrate for RNase Y in vivo and in vitro, transcripts capable of forming the antiterminator were only cleaved in the presence of SAM. Turnover of 10 other SAM-dependent riboswitches was also initiated by RNase Y. Depletion of this ribonuclease increased the half-life of bulk mRNA more than two-fold. This indicates that RNase Y might be not only important for riboswitch RNA turnover but also as a key player in the initiation of mRNA decay in B. subtilis. About 40% of the sequenced eubacterial species have an RNase Y orthologue.

Structural insights into the dual activity of RNase J.

Abstract: The maturation and stability of RNA transcripts is controlled by a combination of endo- and exoRNases. RNase J is unique, as it combines an RNase E–like endoribonucleolytic and a 5′-to-3′ exoribonucleolytic activity in a single polypeptide. The structural basis for this dual activity is unknown. Here we report the crystal structures of Thermus thermophilus RNase J and its complex with uridine 5′-monophosphate. A binding pocket coordinating the phosphate and base moieties of the nucleotide in the vicinity of the catalytic center provide a rationale for the 5′-monophosphate–dependent 5′-to-3′ exoribonucleolytic activity. We show that this dependence is strict; an initial 5′-PPP transcript cannot be degraded exonucleolytically from the 5′-end. Our results suggest that RNase J might switch promptly from endo- to exonucleolytic mode on the same RNA, a property that has important implications for RNA metabolism in numerous prokaryotic organisms and plant organelles containing RNase J orthologs.

The phylogenetic distribution of bacterial ribonucleases

Abstract: Ribonucleases play key, often essential, roles in cellular metabolism. Nineteen ribonuclease activities, from 22 different proteins, have so far been described in bacteria, the majority of them from either Escherichia coli or Bacillus subtilis. Here we examine the phylogenetic distribution of all of these ribonucleases in 50 eubacterial and archaeal species whose genomes have been completely sequenced, with particular emphasis on the endoribonucleases. Although some enzymes are very highly conserved throughout evolution, there appears to be no truly universal ribonuclease. While some organisms, like E.coli, have a large selection of ribonucleases, many with overlapping functions, others seem to have relatively few or have many that remain to be discovered.

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis.

Abstract: In contrast to Escherichia coli , where the 3′ ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3′ end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3′ tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3′ to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis . Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo , none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro . We have thus renamed the gene rnz . A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro , with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5′ extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5′ end by RNase P may have to precede RNase Z cleavage.

Integrative Genomics Viewer

Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

Abstract: The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

Simple method for identification of plasmid-coded proteins. [Escherichia coli, uv radiation]

Abstract: Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.