RNA-structural Mimicry in Escherichia coli Ribosomal Protein L4-dependent Regulation of the S10 Operon

Abstract

Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination.Ribosomal proteins are encoded on the bacterial chromosomes in multigene operons that facilitate stoichiometric production of the more than 50 ribosomal proteins (r-proteins). 1Expression of most of these operons is translationally regulated by a single regulatory r-protein encoded by a given polycistronic mRNA. This operon-specific autogenous control mechanism is elicited when repressor r-proteins are not consumed during the assembly of ribosomal subunits. In the absence of sufficient ribosomal RNA targets, the resulting excess of "free" repressor proteins bind to their own mRNAs and block translation of the polycistronic mRNA (reviewed in Refs. 1 and 2).R-protein L4 from Escherichia coli specifically regulates the S10 operon, which codes for 11 r-proteins including L4 itself (3, 4). L4 is unique among the regulatory ribosomal proteins because it regulates not only translation but also transcription of the S10 operon mRNA. The latter form of regulation is accomplished by L4 stimulation of transcription termination at a terminator (attenuator) structure in the mRNA upstream of the initiation codon of the first gene of the operon (5). Determinants necessary for L4-mediated autogenous control are contained within a 172-nucleotide 5Ј-untranslated region of the S10 operon mRNA. This region folds into six stem-loop structures termed helices HA, HB, HC, HD, HE, and HG (Fig. 1A) (6, 7).The mRNA sequences necessary for L4-mediated transcription and translation control overlap, but are not identical (8, 9). Helix HE and the unstructured sequence immediately downstream of this hairpin are necessary for translational control, whereas helices HD and HE are required for transcriptional control (8, 10, 11). L4-stimulated transcription termination occurs within the U cluster (nt 139 -149) (9) in helix HE, which resembles a Rho-independent terminator. In vitro transcription experiments (10, 12) suggest that the transcription attenuation process i...

A new regulatory circuit in ribosomal protein operons: S2-mediated control of the rpsB-tsf expression in vivo

“…First, the S2 binding to the 59-UTR promoted by S1 may provoke a rearrangement of the mRNA leader structure inhibitory for translation-initiation complex formation; in this case, translational repression of the rpsB mRNA may exert a polar effect on the tsf expression via breaking the transcription-translation coupling within rpsB. Alternatively, the S2-mRNA complex itself or in concert with other factors may directly stimulate preliminary transcription termination, as in the case of the L4-mediated repression of the S10 operon (Zengel and Lindahl 1994;Stelzl et al 2003). Future experiments (mainly in vitro) will shed light upon the fine mechanism underlying the S2-mediated negative control of the rpsB-tsf expression.…”

“…Most of the primary r-proteins acting as autogenous regulators (e.g., S7, S8, S15, L1, L4, L20) recognize similar targets on free rRNA and on mRNA of its cognate operon (Zengel and Lindahl 1994;Guillier et al 2002Guillier et al , 2005Stelzl et al 2003;Mathy et al 2004), indicating that the autogenous control is based on molecular mimicry as was originally proposed by Nomura (Nomura et al 1980). A detailed analysis of the refined crystal structure of the T. thermophilus 30S subunit at 3.05Å resolution revealed that S2 is located on the back of 30S and spans the head-body hinge region by forming contacts with 16S rRNA helices H26 in the body and H35-H37 in the head of the subunit (Brodersen et al 2002).…”

Regulation of Ribosomal Protein Synthesis in Vibrio cholerae

“…Hairpin HD is required for efficient L4 control of transcription in E. coli (29) and is essential for binding of L4 to the S10 leader in vitro (20), but deletion of HD has little effect on translation (29). We deleted hairpin HD from the V. cholerae leader and observed significantly reduced inhibition of expression of the fusion protein (Fig.…”

Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria

“…This hairpin seems to be required only for the L25-mediated control but not for translation efficiency, since its deletions increased the translation yield, simultaneously decreasing autogenous repression. At first glance, a set of base parings within HII does not resemble the L25 target on 5S rRNA, the loop E, so that at present it is hard to suggest (or to rule out) that binding of L25 to its mRNA mimics its interaction with 5S rRNA, following the principle of "molecular mimicry" earlier proposed for a number of r-protein operons (Nomura et al 1980;Stelzl et al 2003;Mathy et al 2004;Guillier et al 2005;Fu et al 2014).…”