Artykuły w czasopismach: „Anti-Shine Dalgarno”

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Weiner, Iddo, Noam Shahar, Pini Marco, Iftach Yacoby i Tamir Tuller. "Solving the Riddle of the Evolution of Shine-Dalgarno Based Translation in Chloroplasts". Molecular Biology and Evolution 36, nr 12 (10.09.2019): 2854–60. http://dx.doi.org/10.1093/molbev/msz210.

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Abstract Chloroplasts originated from an ancient cyanobacterium and still harbor a bacterial-like genome. However, the centrality of Shine–Dalgarno ribosome binding, which predominantly regulates proteobacterial translation initiation, is significantly decreased in chloroplasts. As plastid ribosomal RNA anti-Shine–Dalgarno elements are similar to their bacterial counterparts, these sites alone cannot explain this decline. By computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine–Dalgarno locus, which in-turn reduce the probability of ribosome binding. To validate our model, we expressed two reporter genes (mCherry, hydrogenase) harboring a Shine–Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. Coexpressing them with a 16S ribosomal RNA, modified according to our model, significantly enhances mCherry and hydrogenase expression compared with coexpression with an endogenous 16S gene.

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Stower, Hannah. "Anti-Shine–Dalgarno regulation of translation". Nature Reviews Genetics 13, nr 5 (12.04.2012): 298. http://dx.doi.org/10.1038/nrg3233.

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Praszkier, J., i A. J. Pittard. "Pseudoknot-Dependent Translational Coupling in repBA Genes of the IncB Plasmid pMU720 Involves Reinitiation". Journal of Bacteriology 184, nr 20 (15.10.2002): 5772–80. http://dx.doi.org/10.1128/jb.184.20.5772-5780.2002.

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ABSTRACT Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

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Amin, Mohammad Ruhul, Alisa Yurovsky, Yuping Chen, Steve Skiena i Bruce Futcher. "Re-annotation of 12,495 prokaryotic 16S rRNA 3’ ends and analysis of Shine-Dalgarno and anti-Shine-Dalgarno sequences". PLOS ONE 13, nr 8 (23.08.2018): e0202767. http://dx.doi.org/10.1371/journal.pone.0202767.

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Li, Gene-Wei, Eugene Oh i Jonathan S. Weissman. "The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria". Nature 484, nr 7395 (28.03.2012): 538–41. http://dx.doi.org/10.1038/nature10965.

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Petropoulos, Alexandros D., i Rachel Green. "Further in Vitro Exploration Fails to Support the Allosteric Three-site Model". Journal of Biological Chemistry 287, nr 15 (29.02.2012): 11642–48. http://dx.doi.org/10.1074/jbc.c111.330068.

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Ongoing debate in the ribosome field has focused on the role of bound E-site tRNA and the Shine-Dalgarno-anti-Shine-Dalgarno (SD-aSD) interaction on A-site tRNA interactions and the fidelity of tRNA selection. Here we use an in vitro reconstituted Escherichia coli translation system to explore the reported effects of E-site-bound tRNA and SD-aSD interactions on tRNA selection events and find no evidence for allosteric coupling. A large set of experiments exploring the role of the E-site tRNA in miscoding failed to recapitulate the observations of earlier studies (Di Giacco, V., Márquez, V., Qin, Y., Pech, M., Triana-Alonso, F. J., Wilson, D. N., and Nierhaus, K. H. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 10715–10720 and Geigenmüller, U., and Nierhaus, K. H. (1990) EMBO J. 9, 4527–4533); the frequency of miscoding was unaffected by the presence of E-site-bound cognate tRNA. Moreover, our data provide clear evidence that the reported effects of the SD-aSD interaction on fidelity can be attributed to the binding of ribosomes to an unanticipated site on the mRNA (in the absence of the SD sequence) that provides a cognate pairing codon leading naturally to incorporation of the purported “noncognate” amino acid.

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Moll, Isabella, Michael Huber, Sonja Grill, Pooneh Sairafi, Florian Mueller, Richard Brimacombe, Paola Londei i Udo Bläsi. "Evidence against an Interaction between the mRNA Downstream Box and 16S rRNA in Translation Initiation". Journal of Bacteriology 183, nr 11 (1.06.2001): 3499–505. http://dx.doi.org/10.1128/jb.183.11.3499-3505.2001.

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ABSTRACT Based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mRNAs to bases 1469 to 1483 in helix 44 of 16S rRNA (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the Shine-Dalgarno (SD)/anti-Shine-Dalgarno (aSD) interaction. Computer modeling of helix 44 on the 30S subunit shows that the topography of the 30S ribosome does not allow a simultaneous db-adb interaction and placement of the initiation codon in the ribosomal P site. Thus, the db-adb interaction cannot substitute for the SD-aSD interaction in translation initiation. We have always argued that any contribution of the db-adb interaction should be most apparent on mRNAs devoid of an SD sequence. Here, we show that 30S ribosomes do not bind to leaderless mRNA in the absence of initiator tRNA, even when the initial coding region shows a 15-nucleotide complementarity (optimal fit) with the putative adb. In addition, an optimized db did not affect the translational efficiency of a leaderless λ cI-lacZ reporter construct. Thus, the db-adb interaction can hardly serve as an initial recruitment signal for ribosomes. Moreover, we show that different leaderless mRNAs are translated in heterologous systems although the sequence of the putative adb's within helix 44 of the 30S subunits of the corresponding bacteria differ largely. Taken our data together with those of others (M. O'Connor, T. Asai, C. L. Squires, and A. E. Dahlberg, Proc. Natl. Acad. Sci. USA 96:8973–8978, 1999; A. La Teana, A. Brandi, M. O'Connor, S. Freddi, and C. L. Pon, RNA 6:1393–1402, 2000), we conclude that the db does not base pair with the adb.

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Poot, Raymond A., Marcel F. Brink, Cornelis W. A. Pleji, Herman A. de Boer i Jan Van Duin. "Separation of mutant and wild-type ribosomes based on differences in their anti Shine - Dalgarno sequence". Nucleic Acids Research 21, nr 23 (1993): 5398–402. http://dx.doi.org/10.1093/nar/21.23.5398.

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Stefan, Alessandra, Flavio Schwarz, Daniela Bressanin i Alejandro Hochkoeppler. "Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli". Journal of Bioscience and Bioengineering 110, nr 5 (listopad 2010): 523–28. http://dx.doi.org/10.1016/j.jbiosc.2010.05.012.

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Skorski, Patricia, Prune Leroy, Olivier Fayet, Marc Dreyfus i Sylvie Hermann-Le Denmat. "The Highly Efficient Translation Initiation Region from the Escherichia coli rpsA Gene Lacks a Shine-Dalgarno Element". Journal of Bacteriology 188, nr 17 (1.09.2006): 6277–85. http://dx.doi.org/10.1128/jb.00591-06.

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ABSTRACT The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position −91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the “specialized ribosome” approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.

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Hockenberry, Adam J., Adam R. Pah, Michael C. Jewett i Luís A. N. Amaral. "Leveraging genome-wide datasets to quantify the functional role of the anti-Shine–Dalgarno sequence in regulating translation efficiency". Open Biology 7, nr 1 (styczeń 2017): 160239. http://dx.doi.org/10.1098/rsob.160239.

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Studies dating back to the 1970s established that sequence complementarity between the anti-Shine–Dalgarno (aSD) sequence on prokaryotic ribosomes and the 5′ untranslated region of mRNAs helps to facilitate translation initiation. The optimal location of aSD sequence binding relative to the start codon, the full extents of the aSD sequence and the functional form of the relationship between aSD sequence complementarity and translation efficiency have not been fully resolved. Here, we investigate these relationships by leveraging the sequence diversity of endogenous genes and recently available genome-wide estimates of translation efficiency. We show that—after accounting for predicted mRNA structure—aSD sequence complementarity increases the translation of endogenous mRNAs by roughly 50%. Further, we observe that this relationship is nonlinear, with translation efficiency maximized for mRNAs with intermediate levels of aSD sequence complementarity. The mechanistic insights that we observe are highly robust: we find nearly identical results in multiple datasets spanning three distantly related bacteria. Further, we verify our main conclusions by re-analysing a controlled experimental dataset.

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Vasquez, Kevin A., Taylor A. Hatridge, Nicholas C. Curtis i Lydia M. Contreras. "Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine–Dalgarno Sequence Improves Solubility". ACS Synthetic Biology 5, nr 2 (16.12.2015): 133–45. http://dx.doi.org/10.1021/acssynbio.5b00193.

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Nikolaeva, Daria D., Mikhail S. Gelfand i Sofya K. Garushyants. "Simplification of Ribosomes in Bacteria with Tiny Genomes". Molecular Biology and Evolution 38, nr 1 (18.07.2020): 58–66. http://dx.doi.org/10.1093/molbev/msaa184.

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Abstract The ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (<1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be positioned on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of ribosomal RNA is also common, with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine–Dalgarno sequence is associated with the loss of a higher number of ribosomal proteins.

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Wei, Yulong, i Xuhua Xia. "Unique Shine–Dalgarno Sequences in Cyanobacteria and Chloroplasts Reveal Evolutionary Differences in Their Translation Initiation". Genome Biology and Evolution 11, nr 11 (22.10.2019): 3194–206. http://dx.doi.org/10.1093/gbe/evz227.

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Abstract Microorganisms require efficient translation to grow and replicate rapidly, and translation is often rate-limited by initiation. A prominent feature that facilitates translation initiation in bacteria is the Shine–Dalgarno (SD) sequence. However, there is much debate over its conservation in Cyanobacteria and in chloroplasts which presumably originated from endosymbiosis of ancient Cyanobacteria. Elucidating the utilization of SD sequences in Cyanobacteria and in chloroplasts is therefore important to understand whether 1) SD role in Cyanobacterial translation has been reduced prior to chloroplast endosymbiosis or 2) translation in Cyanobacteria and in plastid has been subjected to different evolutionary pressures. To test these alternatives, we employed genomic, proteomic, and transcriptomic data to trace differences in SD usage among Synechocystis species, Microcystis aeruginosa, cyanophages, Nicotiana tabacum chloroplast, and Arabidopsis thaliana chloroplast. We corrected their mis-annotated 16S rRNA 3′ terminus using an RNA-Seq-based approach to determine their SD/anti-SD locational constraints using an improved measurement DtoStart. We found that cyanophages well-mimic Cyanobacteria in SD usage because both have been under the same selection pressure for SD-mediated initiation. Whereas chloroplasts lost this similarity because the need for SD-facilitated initiation has been reduced in plastids having much reduced genome size and different ribosomal proteins as a result of host-symbiont coevolution. Consequently, SD sequence significantly increases protein expression in Cyanobacteria but not in chloroplasts, and only Cyanobacterial genes compensate for a lack of SD sequence by having weaker secondary structures at the 5′ UTR. Our results suggest different evolutionary pressures operate on translation initiation in Cyanobacteria and in chloroplast.

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Baez, William D., Bappaditya Roy, Zakkary A. McNutt, Elan A. Shatoff, Shicheng Chen, Ralf Bundschuh i Kurt Fredrick. "Global analysis of protein synthesis in Flavobacterium johnsoniae reveals the use of Kozak-like sequences in diverse bacteria". Nucleic Acids Research 47, nr 20 (11.10.2019): 10477–88. http://dx.doi.org/10.1093/nar/gkz855.

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Abstract In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine–Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position –3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved β-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.

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Melancon, Pierre, Daniel Leclerc, Nathalie Destroismaisons i Lea Brakier-Gingras. "The anti-Shine-Dalgarno region in Escherichia coli 16S ribosomal RNA is not essential for the correct selection of translational starts". Biochemistry 29, nr 13 (3.04.1990): 3402–7. http://dx.doi.org/10.1021/bi00465a037.

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Carter-Muenchau, P., i R. E. Wolf. "Growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level mediated by an anti-Shine-Dalgarno sequence located within the Escherichia coli gnd structural gene." Proceedings of the National Academy of Sciences 86, nr 4 (1.02.1989): 1138–42. http://dx.doi.org/10.1073/pnas.86.4.1138.

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Tanabe, Yasushi, Takeo Wada, Katsuhiko Ono, Tatsuhiko Abo i Kazuhiro Kutsukake. "The Transcript from the σ28-Dependent Promoter Is Translationally Inert in the Expression of the σ28-Encoding GenefliAin thefliAZOperon of Salmonella enterica Serovar Typhimurium". Journal of Bacteriology 193, nr 22 (9.09.2011): 6132–41. http://dx.doi.org/10.1128/jb.05909-11.

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There are three classes of promoters for flagellar operons inSalmonella. Class 2 promoters are transcribed by σ70RNA polymerase in the presence of an essential activator, FlhD4C2, and activated by an auxiliary regulator, FliZ. Class 3 promoters are transcribed by σ28RNA polymerase and repressed by an anti-σ28factor, FlgM. σ28(FliA) and FliZ are encoded by thefliAandfliZgenes, respectively, which together constitute an operon transcribed in this order. This operon is transcribed from both class 2 and class 3 promoters, suggesting that it should be activated by its own product, σ28, even in the absence of FlhD4C2. However, σ28-dependent transcription occursin vivoonly in the presence of FlhD4C2, indicating that transcription from the class 2 promoter is a prerequisite to that from the class 3 promoter. In this study, we examined the effects of variously modified versions of thefliAregulatory region on transcription and translation of thefliAgene. We showed that FliA is not significantly translated from the class 3 transcript. In contrast, the 5′-terminal AU-rich sequence found in the class 2 transcript confers efficientfliAtranslation. Replacement of the Shine-Dalgarno sequence of thefliAgene with a better one improvedfliAtranslation from the class 3 transcript. These results suggest that the 5′-terminal AU-rich sequence of the class 2 transcript may assist ribosome binding. FliZ was shown to be expressed from both the class 2 and class 3 transcripts.

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Levitin, Anastasia, i Charles Yanofsky. "Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and trp Operon Expression in Bacillus licheniformis". Journal of Bacteriology 192, nr 6 (8.01.2010): 1518–26. http://dx.doi.org/10.1128/jb.01420-09.

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ABSTRACT Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNATrp. Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNATrp deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis.

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Dutta, Debapratim, i Joseph E. Wedekind. "Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation". Journal of Biological Chemistry 295, nr 9 (28.10.2019): 2555–67. http://dx.doi.org/10.1074/jbc.ra119.010755.

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Riboswitches are a class of nonprotein-coding RNAs that directly sense cellular metabolites to regulate gene expression. They are model systems for analyzing RNA-ligand interactions and are established targets for antibacterial agents. Many studies have analyzed the ligand-binding properties of riboswitches, but this work has outpaced our understanding of the underlying chemical pathways that govern riboswitch-controlled gene expression. To address this knowledge gap, we prepared 15 mutants of the preQ1-II riboswitch—a structurally and biochemically well-characterized HLout pseudoknot that recognizes the metabolite prequeuosine1 (preQ1). The mutants span the preQ1-binding pocket through the adjoining Shine–Dalgarno sequence (SDS) and include A-minor motifs, pseudoknot-insertion helix P4, U·A-U base triples, and canonical G-C pairs in the anti-SDS. As predicted—and confirmed by in vitro isothermal titration calorimetry measurements—specific mutations ablated preQ1 binding, but most aberrant binding effects were corrected by compensatory mutations. In contrast, functional analysis in live bacteria using a riboswitch-controlled GFPuv-reporter assay revealed that each mutant had a deleterious effect on gene regulation, even when compensatory changes were included. Our results indicate that effector binding can be uncoupled from gene regulation. We attribute loss of function to defects in a chemical interaction network that links effector binding to distal regions of the fold that support the gene-off RNA conformation. Our findings differentiate effector binding from biological function, which has ramifications for riboswitch characterization. Our results are considered in the context of synthetic ligands and drugs that bind tightly to riboswitches without eliciting a biological response.

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Dennerlein, Sven, Agata Rozanska, Mateusz Wydro, Zofia M. A. Chrzanowska-Lightowlers i Robert N. Lightowlers. "Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit". Biochemical Journal 430, nr 3 (27.08.2010): 551–58. http://dx.doi.org/10.1042/bj20100757.

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The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit [12S mt (mitochondrial)-rRNA]. Bacterial Era associates with a 3′ unstructured nonanucleotide immediately downstream of the terminal stem–loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine–Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3′ terminal stem–loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA.

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Xia, Xuhua. "Optimizing Protein Production in Therapeutic Phages against a Bacterial Pathogen, Mycobacterium abscessus". Drugs and Drug Candidates 2, nr 1 (21.03.2023): 189–209. http://dx.doi.org/10.3390/ddc2010012.

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Therapeutic phages against pathogenic bacteria should kill the bacteria efficiently before the latter evolve resistance against the phages. While many factors contribute to phage efficiency in killing bacteria, such as phage attachment to host, delivery of phage genome into the host, phage mechanisms against host defense, phage biosynthesis rate, and phage life cycle, this paper focuses only on the optimization of phage mRNA for efficient translation. Phage mRNA may not be adapted to its host translation machinery for three reasons: (1) mutation disrupting adaptation, (2) a recent host switch leaving no time for adaptation, and (3) multiple hosts with different translation machineries so that adaptation to one host implies suboptimal adaptation to another host. It is therefore important to optimize phage mRNAs in therapeutic phages. Theoretical and practical principles based on many experiments were developed and applied to phages engineered against a drug-resistant Mycobacterium abscessus that infected a young cystic fibrosis patient. I provide a detailed genomic evaluation of the three therapeutic phages with respect to translation initiation, elongation, and termination, by making use of both experimental results and highly expressed genes in the host. For optimizing phage genes against M. abscessus, the start codon should be AUG. The DtoStart distance from base-pairing between the Shine-Dalgarno (SD) sequence and the anti-SD sequence should be 14–16. The stop codon should be UAA. If UAG or UGA is used as a stop codon, they should be followed by nucleotide U. Start codon, SD, or stop codon should not be embedded in a secondary structure that may obscure the signals and interfere with their decoding. The optimization framework should be generally applicable to developing therapeutic phages against bacterial pathogens.

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Saito, Kazuki, Rachel Green i Allen R. Buskirk. "Translational initiation in E. coli occurs at the correct sites genome-wide in the absence of mRNA-rRNA base-pairing". eLife 9 (17.02.2020). http://dx.doi.org/10.7554/elife.55002.

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Shine-Dalgarno (SD) motifs are thought to play an important role in translational initiation in bacteria. Paradoxically, ribosome profiling studies in E. coli show no correlation between the strength of an mRNA’s SD motif and how efficiently it is translated. Performing profiling on ribosomes with altered anti-Shine-Dalgarno sequences, we reveal a genome-wide correlation between SD strength and ribosome occupancy that was previously masked by other contributing factors. Using the antibiotic retapamulin to trap initiation complexes at start codons, we find that the mutant ribosomes select start sites correctly, arguing that start sites are hard-wired for initiation through the action of other mRNA features. We show that A-rich sequences upstream of start codons promote initiation. Taken together, our genome-wide study reveals that SD motifs are not necessary for ribosomes to determine where initiation occurs, though they do affect how efficiently initiation occurs.

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McNutt, Zakkary A., Mai D. Gandhi, Elan A. Shatoff, Bappaditya Roy, Aishwarya Devaraj, Ralf Bundschuh i Kurt Fredrick. "Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli". Frontiers in Molecular Biosciences 8 (13.12.2021). http://dx.doi.org/10.3389/fmolb.2021.787388.

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The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms.

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Jha, Vikash, Bappaditya Roy, Dushyant Jahagirdar, Zakkary A. McNutt, Elan A. Shatoff, Bethany L. Boleratz, Dean E. Watkins i in. "Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome". Nucleic Acids Research, 16.12.2020. http://dx.doi.org/10.1093/nar/gkaa1195.

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Abstract Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein—bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.

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Bartoli, J., L. My, Lucid Belmudes, Yohann Couté, J. P. Viala i E. Bouveret. "The Long Hunt for pssR—Looking for a Phospholipid Synthesis Transcriptional Regulator, Finding the Ribosome". Journal of Bacteriology 199, nr 14 (8.05.2017). http://dx.doi.org/10.1128/jb.00202-17.

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ABSTRACT The phospholipid (PL) composition of bacterial membranes varies as a function of growth rate and in response to changes in the environment. While growth adaptation can be explained by biochemical feedback in the PL synthesis pathway, recent transcriptome studies have revealed that the expression of PL synthesis genes can also be tuned in response to various stresses. We previously showed that the BasRS two-component pathway controls the expression of the diacylglycerol kinase gene, dgkA, in Escherichia coli (A. Wahl, L. My, R. Dumoulin, J. N. Sturgis, and E. Bouveret, Mol Microbiol, 80:1260–1275, 2011, https://doi.org/10.1111/j.1365-2958.2011.07641.x ). In this study, we set up a strategy to identify the mutation responsible for the upregulation of pssA observed in the historical pssR1 mutant and supposedly corresponding to a transcriptional repressor (C. P. Sparrow and J. Raetz, J Biol Chem, 258:9963–9967, 1983). pssA encodes phosphatidylserine synthase, the first step of phosphatidylethanolamine synthesis. We showed that this mutation corresponded to a single nucleotide change in the anti-Shine-Dalgarno sequence of the 16S rRNA encoded by the rrnC operon. We further demonstrated that this mutation enhanced the translation of pssA. Though this effect appeared to be restricted to PssA among phospholipid synthesis enzymes, it was not specific, as evidenced by a global effect on the production of unrelated proteins. IMPORTANCE Bacteria adjust the phospholipid composition of their membranes to the changing environment. In addition to enzymatic regulation, stress response regulators control specific steps of the phospholipid synthesis pathway. We wanted to identify a potential regulator controlling the expression of the phosphatidylserine synthase gene. We showed that it was not the previously suggested hdfR gene and instead that a mutation in the anti-Shine-Dalgarno sequence of 16S RNA was responsible for an increase in pssA translation. This example underlines the fact that gene expression can be modulated by means other than specific regulatory processes.

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Metelev, Mikhail, Erik Lundin, Ivan L. Volkov, Arvid H. Gynnå, Johan Elf i Magnus Johansson. "Direct measurements of mRNA translation kinetics in living cells". Nature Communications 13, nr 1 (6.04.2022). http://dx.doi.org/10.1038/s41467-022-29515-x.

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AbstractRibosome mediated mRNA translation is central to life. The cycle of translation, however, has been characterized mostly using reconstituted systems, with only few techniques applicable for studies in the living cell. Here we describe a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding and translating an mRNA, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in translation at any particular time, and that the 30S and 50S ribosomal subunits spend the same average time bound to an mRNA, revealing that 30S re-initiation on poly-cistronic mRNAs is not prevalent in E. coli. Instead, our results are best explained by substantial 70S re-initiation of translation of poly-cistronic mRNAs, which is further corroborated by experiments with translation initiation inhibitors. Finally, we find that a variety of previously described orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs.

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Silke, Jordan R., Yulong Wei i Xuhua Xia. "RNA-Seq-Based Analysis Reveals Heterogeneity in Mature 16S rRNA 3' Termini and Extended Anti-Shine-Dalgarno Motifs in Bacterial Species". G3: Genes|Genomes|Genetics, 24.10.2018, g3.200729.2018. http://dx.doi.org/10.1534/g3.118.200729.

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