scholarly journals Saturation mutagenesis of a +1 programmed frameshift-inducing mRNA sequence derived from a yeast retrotransposon

RNA ◽  
2007 ◽  
Vol 13 (11) ◽  
pp. 1940-1947 ◽  
Author(s):  
C. Guarraia ◽  
L. Norris ◽  
A. Raman ◽  
P. J. Farabaugh
2018 ◽  
Vol 34 (6) ◽  
pp. 33-42
Author(s):  
T.L. Gordeeva ◽  
◽  
L.N. Borshchevskaya ◽  
A.N. Kalinina ◽  
S.P. Sineoky ◽  
...  

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 945
Author(s):  
Christophe Delehedde ◽  
Luc Even ◽  
Patrick Midoux ◽  
Chantal Pichon ◽  
Federico Perche

Messenger RNA (mRNA) is being extensively used in gene therapy and vaccination due to its safety over DNA, in the following ways: its lack of integration risk, cytoplasmic expression, and transient expression compatible with fine regulations. However, clinical applications of mRNA are limited by its fast degradation by nucleases, and the activation of detrimental immune responses. Advances in mRNA applications, with the recent approval of COVID-19 vaccines, were fueled by optimization of the mRNA sequence and the development of mRNA delivery systems. Although delivery systems and mRNA sequence optimization have been abundantly reviewed, understanding of the intracellular processing of mRNA is mandatory to improve its applications. We will focus on lipid nanoparticles (LNPs) as they are the most advanced nanocarriers for the delivery of mRNA. Here, we will review how mRNA therapeutic potency can be affected by its interactions with cellular proteins and intracellular distribution.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shin Irumagawa ◽  
Kaito Kobayashi ◽  
Yutaka Saito ◽  
Takeshi Miyata ◽  
Mitsuo Umetsu ◽  
...  

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.


Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.


Nature ◽  
2021 ◽  
Author(s):  
Ferran Muiños ◽  
Francisco Martínez-Jiménez ◽  
Oriol Pich ◽  
Abel Gonzalez-Perez ◽  
Nuria Lopez-Bigas

Author(s):  
Christoph Öhlknecht ◽  
Sonja Katz ◽  
Christina Kröß ◽  
Bernhard Sprenger ◽  
Petra Engele ◽  
...  

Catalysts ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 665
Author(s):  
Li Zong ◽  
Yan Zhang ◽  
Zhengkang Shao ◽  
Yingwu Wang ◽  
Zheng Guo ◽  
...  

Cytochrome P450 (CYP) mediated enzymatic hydroxylation of fatty acids present a green alternative to chemical synthesis of hydroxy fatty acids (HFAs), which are high-value oleochemicals with various uses in materials industry and medical field. Although many CYPs require the presence of additional reductase proteins for catalytic activity, self-sufficient CYPs have their reductase partner naturally fused into their catalytic domain, leading to a greatly simplified biotransformation process. A recently discovered self-sufficient CYP, BAMF2522 from Bacillus amyloliquefaciens DSM 7, exhibits novel regioselectivity by hydroxylating in-chain positions of palmitic acid generating ω-1 to ω-7 HFAs, a rare regiodiversity profile among CYPs. Besides, F89I mutant of BAMF2522 expanded hydroxylation up to ω-9 position of palmitic acid. Here, we further characterize this enzyme by determining optimum temperature and pH as well as thermal stability. Moreover, using extensive site-directed and site-saturation mutagenesis, we obtained BAMF2522 variants that demonstrate greatly increased regioselectivity for in-chain positions (ω-4 to ω-9) of various medium to long chain fatty acids. Remarkably, when a six-residue mutant was reacted with palmitic acid, 84% of total product content was the sum of ω-7, ω-8 and ω-9 HFA products, the highest in-chain selectivity observed to date with a self-sufficient CYP. In short, our study demonstrates the potential of a recently identified CYP and its mutants for green and sustainable production of a variety of in-chain hydroxy enriched HFAs.


2009 ◽  
Vol 351 (18) ◽  
pp. 3287-3305 ◽  
Author(s):  
Despina J. Bougioukou ◽  
Sabrina Kille ◽  
Andreas Taglieber ◽  
Manfred T. Reetz

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1055-1059 ◽  
Author(s):  
Irina Groisman ◽  
Hanna Engelberg-Kulka

The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described. An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting. Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR–lac′Z fusion. The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study. Using a trpR+1–lac′Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3′ side of the gap is required for bypassing to occur. The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5′ and of Escherichia coli 16S rRNA. A model of trpR+1–lac′Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5′ end of the 16S rRNA and two sites in the mRNA. Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate. The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code.Key words: genetic code, translation, frameshifting, trpR.


Sign in / Sign up

Export Citation Format

Share Document