Transcriptional Regulation of theEscherichia coliGenerraB, Encoding a Protein Inhibitor of RNase E

Abstract

The Escherichia coli RNA degradosome is a protein complex that plays a critical role in the turnover of numerous RNAs. The key component of the degradosome complex is the endoribonuclease RNase E, a multidomain protein composed of an N-terminal catalytic region and a C-terminal region that organizes the other protein components of the degradosome. Previously, the RNase E inhibitors RraA and RraB were identified genetically and shown to bind to the C-terminal region of RNase E, thus affecting both the protein composition of the degradosome and the endonucleolytic activity of RNase E. In the present work, we investigated the transcriptional regulation of rraB. rraB was shown to be transcribed constitutively from its own promoter, PrraB. Transposon mutagenesis and screening for increased ␤-galactosidase activity from a chromosomal PrraB-lacZ transcriptional fusion resulted in the isolation of a transposon insertion in glmS, encoding the essential enzyme glucosamine-6-phosphate synthase that catalyzes the first committed step of the uridine 5-diphospho-N-acetyl-glucosamine (UDP-GlcNAc) pathway, which provides intermediates for peptidoglycan biogenesis. The glmS852::Tn5 allele resulted in an approximately 50% lower intracellular concentration of UDP-GlcNAc and conferred a fivefold increase in the level of rraB mRNA. This allele also mediated a twofold increase in ␤-galactosidase activity from a chromosomal fusion of the 5 untranslated region of the rne gene to lacZ, suggesting that a reduction in cellular concentration of UDP-GlcNAc and the resulting increased expression of RraB might modulate the action of RNase E.The endoribonuclease RNase E plays a central role in RNA metabolism, including the processing of rRNAs and tRNAs (30,42,43); the degradation of small regulatory RNAs; and, most importantly, the turnover of numerous cellular mRNAs in Escherichia coli (5,13,23,33). Homologous RNase E has been identified in more than 50 bacteria, archaea, and plants (28). The 1,061-amino-acid E. coli RNase E protein can be divided into functional portions, from the N terminus to the C terminus, as follows. The N-terminal half (amino acid residues 1 to 529) contains the endonuclease active site (amino acid residues 1 to 395) and a zinc finger region (amino acid residues 400 to 415) (8, 9). The central region includes the membrane anchoring segment A (amino acid residues 565 to 582) and its flanking portions (25) as well as an arginine-rich RNA binding site (amino acid residues 604 to 688) (34). The C-terminal half (amino acid residues 734 to 1061) is an unstructured scaffold domain that contains binding sites for the other core degradosome components, namely, polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase (11,35,45,46,53). Previous studies indicate that the assembled degradosome complex is necessary for normal mRNA degradation and the degradosome components functionally interact during decay of at least some RNAs in E. coli (4,24).The cellular level and activity of RNase E in E. coli are und...

The proteomic response to mutants of the Escherichia coli RNA degradosome

“…This result is also consistent with our recent study in which we searched for mutants which can up-regulate rraB transcription. In our study, we isolated a glmS mutant in which a Tn5 insertion disrupts the coding region of the GlmS protein 22 . RraB disrupts PNPase associated in RNA degradosome both in vivo 23 and in vitro (L. Zhou, G. Georgiou, unpublished ).…”

Structural elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA degradosome

“…To our knowledge, this is the first viral protein identified which uses a direct interaction with the RNA degradosome to inhibit its function. Therefore, Dip can be considered as a viral functional equivalent of the bacterial RraA and RraB (‘regulators of RNase activity’) proteins of E. coli , which inhibit the activity of RNase E by binding to specific regions of the CTH ( Górna et al, 2010 ; Zhou et al, 2009 ).

10.7554/eLife.16413.022 Figure 8.

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“…Moreover, the CTH contains two arginine-rich regions that have been shown to bind RNA: the RNA binding domain (RBD)/Arginine-rich region 1 (AR1) and AR2 ( Callaghan et al, 2004 ). The activity and specificity of the RNA degradosome is under complex regulation and is influenced by several factors including riboregulation by small, non-coding RNAs (sRNA) and proteins RraA and RraB (‘Regulators of RNase Activity A and B’) that inhibit the activity of RNase E by binding to the CTH ( Górna et al, 2010 ; Ikeda et al, 2011 ; Zhou et al, 2009 ).…”

A novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs

“…However, RNase activity is highly dynamic and is regulated by a number of trans-acting regulators, including YmdB, RraA, RraB, as well as other asyet uncharacterized proteins. 17,18,21,22 How broadly the known trans-acting regulators participate in RNA cleavage or decay and whether the regulator-target pairs differ depending on environmental stress are open questions. For example, osmotic regulation of bdm mRNA levels in E. coli is partially RNase III-dependent, 17 but the involvement of YmdB, a regulator of RNase III in response to cold stress, 18 in this process has yet to be fully elucidated.…”