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Translation
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Therefore, the information about the secondary structures of the messengers used, before and after mutagenesis within the SD regions, was found especially crucial, since intra-strand base pairing of a ribosome binding site could have a profound influence on its translational efficiency (de Smit and van Duin 1994b). Varying by site-directed mutagenesis the extent of the SD complementarity in the MS2 coat gene, the authors found that mutations reducing the SD complementarity by one or two nucleotides diminished translational efficiency only if ribosome binding was impaired by the structure of the messenger. Surprisingly, in the absence of an inhibitory structure, these mutations had no effect. In other words, a strong SD interaction was compensated for a structured initiation region. The authors considered therefore the translational initiation on a structured ribosome binding site as a competition between intramolecular base pairing of the messenger and binding to a 30S ribosomal subunit. The good SD complementarity provided the ribosome with an increased affinity for its binding site and thereby enhanced its ability to compete against the secondary structure. Remarkably, this function of the SD interaction closely paralleled the RNA-unfolding capacity of the ribosomal S1 protein (de Smit and van Duin 1994b). The quantitative analysis of literature data on the ribosome binding sites was collected in order to support this idea (de Smit and van Duin 1994a). This compilation showed that the efficiency of translation was determined by the overall stability of the structure at the ribosome binding site, whether the initiation codon itself was base paired or not. The structures weaker than −6 kcal/mol usually did not reduce translational efficiency. Below this threshold, all systems showed a tenfold decrease in expression for every −1.4 kcal/mol (de Smit and van Duin 1994a).
Transcriptionally Regulatory Sequences of Phylogenetic Significance
Published in S. K. Dutta, DNA Systematics, 2019
Once an open complex is formed, RNA chain elongation is rapidly initiated in the presence of substrates. The rate of open complex formation (KB) has been measured using an in vitro mixed transcriptional system.37 During step 1, two classes of DNA promoter sequences are recognized, one contains the consensus hexanucleotide T89A89T50A65A65T,00 and is usually situated −6 to −12 nucleotides preceding the transcription starting point.38 The subscripts denote relative frequency of occurrence;39 transcription start is + 1. The second contains the consensus sextamer sequence T85T93G81A61C69A52 located about −35 nucleotides upstream from the site of transcription initiation. The distance between the Pribnow box (TTAACTA) and the sequence at −35 affects promoter strength, with optimal spacing occurring around 17 bp. High level expression in E. coli of genes cloned in plasmids requires proper placement of the promoter upstream. The optimal distances between Shine-Dalgarno’s AGGA sequence in the promoter-ribosome binding site and the initiation codon ATG for eukaryotic and prokaryotic genes have been determined to be between 7 to 11 and some 40 bases, respectively.40 The reason for this difference may well lie in the secondary structure of the template as well as the transcript, the former of which E. coli RNA polymerase must negotiate. Adequate spacing from 16 to 19 bp is apparently needed between these two promoters for efficient initiation. Neither of these two promoter sequences, however, is absolutely necessary for transcription, although very few promoters lack the −10 site. Other means most likely exist for RNA polymerase to bind and to initiate transcription. For E. coli, RNA polymerase also interacts with sequences upstream of specific promoters.41 A considerable amount of information has been obtained about how chemical alteration or mutation of a single base within these prokaryotic promoters can alter their transcriptional activity as much as 100-fold.42–44 A number of artificial promoter sequences have also been chemically synthesized and shown to be functional.
Riboswitches as therapeutic targets: promise of a new era of antibiotics
Published in Expert Opinion on Therapeutic Targets, 2023
Emily Ellinger, Adrien Chauvier, Rosa A. Romero, Yichen Liu, Sujay Ray, Nils G. Walter
One underexplored mechanism for expanding the clinical arsenal of antibiotics is the targeting of riboswitches. Riboswitches are widespread RNA structural motifs found in the 5’ untranslated regions (5’ UTRs) of mRNAs that in bacteria, including many that are pathogenic (Figure 1 and Table 1), function to regulate transcription, translation, or RNA decay [11,12]. In some bacteria, riboswitches control more than 4% of genes, including those coding for many essential cell products [13]. Two major mechanisms riboswitches exploit to regulate gene expression involve conformational changes that either cause bacterial RNA polymerase (RNAP) to end transcription or prevent translation initiation by sequestering the ribosome binding site [14,15]. Since the transcription and translation machineries are highly conserved among bacteria, but riboswitches are absent in mammals, the application of a drug that targets a riboswitch or a riboswitch-protein interface promise to knock out necessary gene expression across bacterial species while leaving human cells unaffected. Conversely, the diversity of RNA sequences of related riboswitches among bacteria may also offer an angle to target specific pathogens, without harming the beneficial microbiome [16,17].
Applications of the CRISPR-Cas system for infectious disease diagnostics
Published in Expert Review of Molecular Diagnostics, 2021
Peipei Li, Li Wang, Junning Yang, Li-Jun Di, Jingjing Li
Pardee et al. developed nucleic acid sequence-based amplification (NASBA)-CRISPR to discriminate American and African strains of Zika virus as well as detect Zika virus from plasma [39]. The sensitivity of this technology can reach 1–3 fM. The principle of this approach is associated with toehold switch sensors and the specific cleavage ability of Cas9 (Figure 1). Toehold switch sensors are synthetic riboregulators that control LacZ translation by binding RNA. In the absence of binding RNA, the hairpin structure of riboregulators impedes LacZ translation through sequestering the start codon and the ribosome-binding site. While the trigger RNA is complemented to the riboregulator, the start codon and the ribosome-binding site are exposed, and the translation of LacZ is initiated. Finally, LacZ catalyses the conversion of a yellow substrate to a purple product. Double-stranded DNA is generated by the NASBA reaction. When CRISPR-Cas9 cleaves dsDNA, the RNA product transcribed from cleaved dsDNA cannot activate the toehold switch sensor due to the lack of a sensor trigger sequence. In their research, they amplified Zika virus RNA using isothermal amplification, targeting the binding RNA of riboregulators.
Mining for missed sORF-encoded peptides
Published in Expert Review of Proteomics, 2019
Xinqiang Yin, Yuanyuan Jing, Hanmei Xu
The combination of large-scale transcriptome analyses and computational biology can be applied to find putative protein-coding sORFs. However, many of these strategies overlook the compositions of the 5´-upstream sequences and 3´-downstream sequences of the sORFs. There are key elements for translation, such as ribosome binding sites, in these untranslated regions. Although Kozak sequence has already been reported, it’s difficult to identify it exactly in a given gene for its diversity and indefinity. To date, the common way to validate whether a given sORF identified by computational methods can be translated is in vitro translation assay, in which the full-length cDNA of a putative transcript is cloned into a vector and then express the constructs in cells, then detect whether there is a product of the sORF by many techniques, such as western blot, immunocytochemistry, and immunoprecipitation. This strategy hinges on the concept that ribosomes can distinguish ncRNAs and coding RNAs. But what we must keep in mind is that it is possible that sequences can be translated in vitro but not in vivo. Conversely, if a transcript does not produce a SEP in vitro, that doesn’t mean that the transcript cannot be translated in vivo.