We use an oligonucleotide library of over 5000 variants together with a synthetic biology approach to study a generic context-dependent silencing phenomenon in E. coli. The observed silencing is strongly associated with the presence of short CT-rich motifs (3-5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We provide evidence using modeling, mutations to the CT-rich motif, and synthetic constructs that encode a non-silenced RBS upstream of the silencing motif, that sequestration of the RBS and subsequent rapid messenger degradation is likely to be the mechanism driving the silencing effect. This sequestration is probably due to binding of the RBS to the upstream CU-rich motifs, which we call anti-Shine-Dalgarno (aSD) motifs. To provide further support for the importance of this mechanism in natural systems, we show bioinformatically that the genomes of mesophilic and psychrophilic bacteria are significantly depleted for the observed aSD motifs within 300 bp of putative Shine-Dalgarno motifs (GA-rich hexamers) as compared with a random control in over 70% of the 591 genomes examined. In contrast, in genomes of thermophilic and hyperthermophilic bacteria there is no such depletion, which is consistent with a weak interaction between the short aSD CU-rich motif and the RBS that is thermodynamically less stable at higher ambient living temperatures. Our findings have important implications for understanding SNP/INDEL mutations in regulatory regions, as well as provide a mechanism for promoter/operon insulation in bacterial genomes.