scholarly journals Orthogonal translation enables heterologous ribosome engineering in E. coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Natalie S. Kolber ◽  
Ranan Fattal ◽  
Sinisa Bratulic ◽  
Gavriela D. Carver ◽  
Ahmed H. Badran
Keyword(s):  
Synthetic Biology ◽  
16S Rrna ◽  
General Framework ◽  
Living Cells ◽  
Rrna Processing ◽  
Ribosome Engineering ◽  
Subunit Exchange ◽  
E Coli ◽  
Ribosome Binding ◽  
Promising Avenue

AbstractThe ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.

scholarly journals Sinorhizobium meliloti YbeY is a zinc-dependent single-strand specific endoribonuclease that plays an important role in 16S ribosomal RNA processing

2019 ◽  
Vol 48 (1) ◽  
pp. 332-348 ◽  
Author(s):  
Vignesh M P Babu ◽  
Siva Sankari ◽  
James A Budnick ◽  
Clayton C Caswell ◽  
Graham C Walker
Keyword(s):  
16S Rrna ◽  
Metal Ion ◽  
Sinorhizobium Meliloti ◽  
Single Strand ◽  
Rrna Processing ◽  
E Coli ◽  
Gamma Proteobacteria ◽  
Ribosomal Rna Processing ◽  
Alpha Proteobacteria ◽  
Endoribonuclease Activity

Abstract Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3′ end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.

Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli

2018 ◽  
Vol 430 (12) ◽  
pp. 1745-1759 ◽  
Author(s):  
Brian A. Smith ◽  
Neha Gupta ◽  
Kevin Denny ◽  
Gloria M. Culver
Keyword(s):  
16S Rrna ◽  
Rrna Processing ◽  
Subunit Assembly ◽  
E Coli ◽  
30S Subunit

Subunit Composition of Ribosome in the yqgF Mutant Is Deficient in pre-16S rRNA Processing of Escherichia coli

2018 ◽  
Vol 28 (4) ◽  
pp. 179-182
Author(s):  
Tatsuaki  Kurata ◽  
Shinobu Nakanishi ◽  
Masayuki Hashimoto ◽  
Masato Taoka ◽  
Toshiaki Isobe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Active Form ◽  
Rrna Processing ◽  
Rnase E ◽  
Rnase Iii ◽  
Primary Transcript ◽  
E Coli ◽  
Copy Numbers ◽  
Inactive Form

<i>Escherichia coli</i> 16S, 23S, and 5S ribosomal RNAs (rRNAs) are transcribed as a single primary transcript, which is subsequently processed into mature rRNAs by several RNases. Three RNases (RNase III, RNase E, and RNase G) were reported to function in processing the 5′-leader of precursor 16S rRNA (pre-16S rRNA). Previously, we showed that a novel essential YqgF is involved in that processing. Here we investigated the ribosome subunits of the <i>yqgF</i><sup>ts</sup> mutant by LC-MS/MS. The mutant ribosome had decreased copy numbers of ribosome protein S1, suggesting that the <i>yqgF</i> gene enables incorporation of ribosomal protein S1 into ribosome by processing of the 5′-end of pre-16S rRNA. The ribosome protein S1 is essential for translation in <i>E. coli</i>; therefore, our results suggest that YqgF converts the inactive form of newly synthesized ribosome into the active form at the final step of ribosome assembly.

QGEM 2012: Building a Chimeric Flagellar Scaffold for Enhanced Bioremediation and Biosynthesis

2018 ◽  
Author(s):  
Beini Wang ◽  
Kevin Chen
Keyword(s):  
Synthetic Biology ◽  
Metal Binding ◽  
Living Cells ◽  
Genetically Engineered ◽  
Additional Component ◽  
Protein Subunits ◽  
E Coli ◽  
Self Assembling ◽  
Enhanced Bioremediation ◽  
Real World Problems

Synthetic biology is a rapidly expanding field that involves designing biological systems and operating them in living cells. The Queen’s Genetically Engineered Machine (QGEM) team competes annually at the International Genetically Engineered Machine (iGEM) competition, which challenges students around the globe to use synthetic biology to solve real-world problems. Last year, the QGEM team sought to improve the efficiency of bioremediation and biosynthesis by modifying the flagella of E. coli. Flagella are whip-like appendages that many organisms and cells use for locomotion. One flagellum is composed of 20 000 self-assembling protein subunits called flagellin, which can be divided into two parts. One part is necessary to form the flagellum polymer, and the second part is an auxiliary domain with no particular function. This domain can be replaced with functional proteins, such as metal-binding proteins for bioremediation or useful enzymes for biosynthesis. With the incorporation of proteins for binding, adhesion, degradation, and synthesis, normal flagella can be transformed into functional appendages that can be useful in many applications. As an additional component of their project, the QGEM team developed dance as a new means of teaching and explaining science, and incorporated it into their presentation at the iGEM competition.

scholarly journals RNase AM, a 5′ to 3′ exonuclease, matures the 5′ end of all three ribosomal RNAs in E. coli

2020 ◽  
Vol 48 (10) ◽  
pp. 5616-5623 ◽  
Author(s):  
Chaitanya Jain
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Rna Metabolism ◽  
23S Rrna ◽  
Rrna Processing ◽  
Ribosomal Rnas ◽  
E Coli ◽  
Processing Step ◽  
Rnase G ◽  
Prior Processing

Abstract Bacterial ribosomal RNAs (rRNAs) are transcribed as precursors and require processing by Ribonucleases (RNases) to generate mature and functional rRNAs. Although the initial steps of rRNA processing in Escherichia coli (E. coli) were described several decades ago, the enzymes responsible for the final steps of 5S and 23S rRNA 5′-end maturation have remained unknown. Here, I show that RNase AM, a recently identified 5′ to 3′ exonuclease, performs the last step of 5S rRNA 5′-end maturation. RNase AM was also found to generate the mature 5′ end of 23S rRNA, subsequent to a newly identified prior processing step. Additionally, RNase AM was found to mature the 5′ end of 16S rRNA, a reaction previously attributed to RNase G. These findings indicate a major role for RNase AM in cellular RNA metabolism and establish a biological role for the first 5′ to 3′ RNA exonuclease identified in E. coli.

scholarly journals Elevated Levels of Era GTPase Improve Growth, 16S rRNA Processing, and 70S Ribosome Assembly ofEscherichia coliLacking Highly Conserved Multifunctional YbeY Endoribonuclease

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Anubrata Ghosal ◽  
Vignesh M. P. Babu ◽  
Graham C. Walker
Keyword(s):  
16S Rrna ◽  
Ribosome Biogenesis ◽  
Growth Defect ◽  
Ribosome Assembly ◽  
Rrna Processing ◽  
Content Type ◽  
E Coli ◽  
New Class ◽  
70S Ribosome

ABSTRACTYbeY is a highly conserved, multifunctional endoribonuclease that plays a significant role in ribosome biogenesis and has several additional roles. Here we show that overexpression of the conserved GTPase Era inEscherichia colipartially suppresses the growth defect of a ΔybeYstrain while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires both the ability of Era to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase R, and RNase PH, suggesting a model for the action of Era. Overexpression ofVibrio choleraeEra similarly partially suppresses the defects of anE. coliΔybeYstrain, indicating that this property of Era is conserved in bacteria other thanE. coli.IMPORTANCEThis work provides insight into the critical, but still incompletely understood, mechanism of processing of theE. coli16S rRNA 3′ terminus. The highly conserved GTPase Era is known to bind to the precursor of the 16S rRNA near its 3′ end. Both the endoribonuclease YbeY, which binds to Era, and four exoribonucleases have been implicated in this 3′-end processing. The results reported here offer additional insights into the role of Era in 16S rRNA 3′-end maturation and into the relationship between the action of the endoribonuclease YbeY and that of the four exoribonucleases. This study also hints at why YbeY is essential only in some bacteria and suggests that YbeY could be a target for a new class of antibiotics in these bacteria.

scholarly journals Overexpression of sgm 5’ UTR mRNA reduces gentamicin resistance in both Escherichia coli and Micromonospora melanosporea cells

2007 ◽  
Vol 59 (4) ◽  
pp. 273-280
Author(s):  
M. Kojic ◽  
Sandra Vojnovic ◽  
Natasa Vukov ◽  
Branka Vasiljevic
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Regulatory Region ◽  
Mrna Sequence ◽  
E Coli ◽  
Ribosome Binding ◽  
Gentamicin Resistance ◽  
A Site

The 16S rRNA methylases are expressed by most of the antibiotic producing bacteria in order to protect themselves against antibiotics by methylation of 16S rRNA at positions which are crucial for their action. The sgm sisomicin-gentamicin resistance gene from Micromonospora zionensis methylates G1405 positioned in the A site of 16S rRNA, which includes a CCGCCC hexanucleotide. The same hexanucleotide is also present 14 nucleotides in front of the ribosome binding site of sgm mRNA. The model proposed for translational regulation of sgm assumes that Sgm binds to this motif, both on 16S rRNA and on the 5? untranslated region (UTR) of its own mRNA. The 5? UTR mRNA sequence was overexpressed on 3?-truncated sgm mRNA, and the effect on gentamicin resistance conferred by Sgm was tested in Escherichia coli and in Micromonospora melanosporea. Overexpression of the sgm mRNA regulatory region decreases the resistance to gentamicin in both E. coli and M. melanosporea. This effect is likely to be due to titration of Sgm molecules by the overexpressed 5? UTR.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

scholarly journals Refactoring of a synthetic raspberry ketone pathway with EcoFlex

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Simon J. Moore ◽  
Yonek B. Hleba ◽  
Sarah Bischoff ◽  
David Bell ◽  
Karen M. Polizzi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Combinatorial Libraries ◽  
Value Added ◽  
Microtiter Plate ◽  
Fine Tuning ◽  
Golden Gate ◽  
E Coli ◽  
Fine Chemical ◽  
Raspberry Ketone

Abstract Background  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results  By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions  Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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