Noncoding RNAs Binding to the Nucleoid Protein HU in Escherichia coli

Abstract

Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli. HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro. We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known. HU, a well-conserved and abundant nucleoid protein in Escherichia coli, is composed of two 9-kDa homologous subunits, ␣ and ␤ (9,18,40,41,48,49). HU showed nonspecific interactions with DNA with micromolar affinity (8, 52) and specific interactions with nicked, cruciform, and kinked DNA with nanomolar affinities (5,29,30,45,52,55,60). HU also showed interactions with tRNAs and dsrA and rpoS mRNA (4, 5).It was suggested that RNA molecules, together with HU and other nucleoid proteins, are involved in maintaining E. coli chromosome structure, although the identities of the RNAs have not been established (44). These and subsequent studies showed that 100-to 300-nucleotide (nt)-long RNA chains are part of the nucleoid (43,44). Treatment of the nucleoid with RNase caused a decondensation with a dramatic decrease in the sedimentation constant of the isolated chromosome from 1600S to approximately 400S to 500S (44, 65). In this work, we followed the idea that some HU-RNA complexes are of importance in nucleoid structure with the aim of identifying the RNAs that bind to HU and then understanding the role of such complexes in the nucleoid.We took the ribonomic approach, also referred to as RNA immunoprecipitation (IP) followed by microarray (Chip) profiling (RIP-Chip) assay, which involves immunoprecipitation of ribonucleoprotein (RNP) complexes with antibodies against specific proteins, extraction of RNA, and hybridization to microarrays (58). The assay helped with global identification of putative endogenous RNA targets of specific proteins in several systems (11,12,24,25,32,33,50,51,53,57,66).We report associations of HU with several RNAs and their identification: a s...

DNABII proteins play a central role in UPEC biofilm structure

“…We envision that these proteins and DNA gain access to the extracellular milieu by passive or active cell lysis or autolysis (Claverys and Havarstein, 2007;Rice and Bayles, 2008). It is intriguing that HU is indispensable for the signal recognition particle (SRP) complex in Grampositive bacteria (Nakamura et al, 1999); recently, it was shown that HU may even associate with the 4.5s RNA of the SRP of E. coli (Macvanin et al, 2012). It is possible that HU could exit the bacterial cell via this system.…”

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“…25 Pettijohn and Hecht in 1974 first suggested that RNA plays a role in maintaining the nucleoid structure in E. coli. 26 Recently, Macvanin et al 27 reported that 2 novel non-coding RNAs bind to the architectural DNA-binding protein HU and affect the nucleoid structure, implying that RNA molecules play an important role in genome organization. Moreover, we expect that some of the asRNAs and intraRNAs may code for small peptides, further increasing the protein-coding potential of genomes.…”

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“…77,78 More recently, they have also been demonstrated to be involved in nucleoid morphology and chromosome formation and maintenance. 79 …”