S -adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the S _MK box translational riboswitch RNA

Abstract

The SMK box is a conserved riboswitch motif found in the 5 untranslated region of metK genes [encoding S-adenosylmethionine (SAM) synthetase] in lactic acid bacteria, including Enterococcus, Streptococcus, and Lactococcus sp. Previous studies showed that this RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine-Dalgarno (SD) sequence by pairing with an anti-SD (ASD) element. A model was proposed in which SAM binding inhibits metK translation by preventing binding of the ribosome to the SD region of the mRNA. In the current work, the addition of SAM was shown to inhibit binding of 30S ribosomal subunits to S MK box RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA, which has a disrupted SD-ASD pairing, was defective in SAM binding and showed no reduction of ribosome binding in the presence of SAM, whereas a compensatory mutation that restored SD-ASD pairing restored the response to SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that S MK box translational repression operates through occlusion of the ribosome binding site and that SAM binding requires the SD-ASD pairing.regulatory RNA ͉ RNA structure ͉ translational control ͉ SAM synthetase M any regulatory mechanisms have been discovered recently in bacteria in which RNA transcripts sense a regulatory signal (1-6). These regulatory RNAs, usually called RNA sensors or riboswitches, act in cis to regulate expression of the downstream coding sequence(s) without a requirement for regulatory proteins. Typically, the regulatory signal is an effector molecule (tRNA, noncoding RNA, or small molecule) that binds the nascent RNA transcript, causing a change in the RNA structure. In many systems of this type, the structural change either causes or prevents formation of the helix of an intrinsic transcriptional terminator (reviewed in 1, 4). Similar structural rearrangements can sequester the ribosome binding site (RBS) to regulate at the level of translation initiation (7-9). Binding of the effector to the RNA can also catalyze self-cleavage of the RNA transcript, as in the glmS system (10). Thermosensor riboswitches, in contrast, have no effector binding domain but simply respond by temperature-dependent modulation of the RNA structure (11-13). Riboswitches generally exhibit conserved sequence and structural elements that are responsible for high specificity for their cognate molecular signal.Many members of the Bacillus/Clostridium group of bacteria use the S box riboswitch system to control expression of genes involved in methionine metabolism (14). Included in this regulon is the metK gene, which encodes S-adenosylmethionine (SAM) synthetase, the enzyme responsible for the synthesis of SAM from methionine and ATP. The S box system uses SAM as the effector molecule both in vivo (15, 16) and in vitro (15, 17, 18), and regulation occurs primarily at the level of premature termi...

Dual-acting riboswitch control of translation initiation and mRNA decay

“…Moreover, the lysC riboswitch modulates gene expression at two different genetic levels, namely translation initiation and mRNA decay. initiation of translation upon ligand binding (17,(28)(29)(30)(31). As expected for most translationally repressed mRNAs, the absence of translating ribosomes can reveal critical cleavage site(s) located in the ORF domain that are prone to nuclease attack, ultimately leading to mRNA degradation (32).…”

Control of Methionine Synthesis and Uptake by MetR and Homocysteine in Streptococcus mutans

“…Homoserine O-acyltransferase (MetA) is under allosteric control by methionine and SAM in E. coli and B. subtilis (2,16). In addition, the synthesis of MetK, the first enzyme of the SAM recycling pathway, is regulated by SAM through a riboswitch mechanism in enterococci and possibly in streptococci (12,13). Whether such control systems modulate the cellular homocysteine pool in S. mutans remains to be determined.…”

A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria

“…SAM-III riboswitches commonly regulate gene expression through occlusion of the ribosome binding site or Shine-Dalgarno (SD) sequence. When SAM is bound, the SD base pairs with an anti-SD sequence to form the aptamer, thus preventing ribosome access (Fuchs et al 2007). The distinctive sequence and secondary structures of SAM-III aptamers yield a tertiary structure and binding pocket that are distinct compared to the other SAM riboswitches previously described (Lu et al 2008).…”