Roles of a ribosome-binding site and mRNA secondary structure in differential expression of Shiga toxin genes

Habib, N F; Jackson, Matthew P.

doi:10.1128/jb.175.3.597-603.1993

Cited by 17 publications

(9 citation statements)

References 47 publications

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“…Analysis of the H A 0 cDNA sequence shows rare codons [a CCT codon at position 2 (Pro), TTG codons at positions 3 and 6 (Leu), and the TGT codon at position 5 (Cys)] (Varenne et al, 1984;Grosjean and Fiers, 1982) immediately after the initiation codon; this may reduce translational-initiation efficiency under some physiological conditions and for some genetic backgrounds, leading to uncoupling between transcription and translation. In other cases, changing codons immediately after the initiation ATG codon has succeeded in improving translation efficiency (Buchler et al, 1990;Habib and Jackson, 1993). In conclusion, we suggest that the differenccs observed in the HA0 expression levels may be due to the codon usage at the initiation of translation.…”

Section: Resultsmentioning

confidence: 64%

The Role of a β Barrel Loop 4 Extension in Modulating the Physical and Functional Properties of Long‐Chain 2‐Hydroxy‐Acid Oxidase Isozymes

Belmouden

Lederer

1996

European Journal of Biochemistry

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Peroxisomal long-chain 2-hydroxy-acid oxidase, an FMN-dependent enzyme, catalyzes the oxidation of a variety of L-2-hydroxy acids into keto acids at the expense of oxygen. We recently reported the cloning and sequencing of its cDNA and the existence of a weakly expressed isozyme [Belmouden, A., Biochem. 214,[17][18][19][20][21][22][23][24][25]. This isozyme, Pz, differs from the major one in having a three-residue insertion, -VRK-, in loop 4 of the p8a8 barrel. In the crystal structures of homologous flavocytochrome b, and glycolate oxidase, the corresponding region of loop 4 is disordered. We now report on the constitutive high-level expression of isozymes PI and p2 in Escherichiu coli under control of the ApL promoter, and on the influence of the E. coli genetic background and the growth medium on the expression level. We describe the properties of isozyme fi2 and compare them with those of pure isoform PI. The visible spectra of the purified enzymes differ in the position of the near-ultraviolet band of the prosthetic group. pH titration studies indicate that the FMN ionizes at N3 at a lower pH than free flavin and that there is a small pK, difference between the isozymes. To our knowledge, the only other known case of a lowered pKa for the protein-bound flavin is that of glycolate oxidase. In the CD spectra of the FMN region, a marked difference between isozymes in the 270-300-nm region appears to be related to the pK, difference for the N3-H bond. Kinetic parameters for a number of substrates and inhibitors are indistinguishable within the limits of experimental error, with the exception of values for kczt, for mandelate (the most active substrate), K,,, for hydroxyhippurate (a new substrate), K, for cinnamate and oxalate, and K', for sulfite. The differences are no larger than twofold. The foregoing comparison between isozymes and P2 shows that the naturally engineered insertion in loop 4 exerts some influence on the flavin spectral properties and the active-site reactivity. Since the corresponding loop 4 regions in the three-dimensional structures of flavocytochrome b, and glycolate oxidase are 1.5-2.0 nm removed from the flavin, it would appear either that loop 4 has a very different conformation in hydroxy-acid oxidase, or that it may interact with the active site due to mobility. Keywords: isozyme ; flavin pK, ; 2-hydroxy acid oxidation; recombinant protein expression.Long-chain 2-hydroxy-acid oxidase (HAO) is an FMN-dependent enzyme which oxidizes L-2-hydroxy acids to keto acids at the expense of oxygen, with formation of hydrogen peroxide. It is an isozyme (isozyme B) of short-chain 2-hydroxy-acid oxidase or glycolate oxidase (isozyme A), which is found in mammals and in plants. Both proteins are members of a family of flavoenzymes that dehydrogenate L-2-hydroxy acids of varying structures. For the dehydrogenase-oxidase subclass, the reduced flavin is reoxidized by oxygen, whereas, for the dehydrogenaseelectron transferase subclass, the prosthetic group is reoxidized by a monoelectronic acceptor.From...

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Section: Resultsmentioning

confidence: 64%

The Role of a β Barrel Loop 4 Extension in Modulating the Physical and Functional Properties of Long‐Chain 2‐Hydroxy‐Acid Oxidase Isozymes

Belmouden

Lederer

1996

European Journal of Biochemistry

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“…While promoters play critical roles in gene regulation, other gene features and mechanisms have additional important regulatory roles. One such important gene feature is the 5’ untranslated region (5’ UTR), which contains the Shine-Dalgarno (SD) sequence within the ribosome binding site (RBS), and therefore can serve as a translation regulator [1-5]. For example, 5’ UTR interactions with cis and trans elements, such as complementary sequences within the UTR or coding sequence, small RNAs (sRNAs), and RNA-binding proteins, can modulate protein synthesis by blocking or improving accessibility to the RBS [6-9].…”

Section: Introductionmentioning

confidence: 99%

The impact of leadered and leaderless gene structures on translation efficiency, transcript stability, and predicted transcription rates inMycobacterium smegmatis

Nguyen

Vargas-Blanco

Roberts

et al. 2019

Preprint

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16Regulation of gene expression is critical for the pathogen Mycobacterium tuberculosis to tolerate 17 stressors encountered during infection, and for non-pathogenic mycobacteria such as 18Mycobacterium smegmatis to survive stressors encountered in the environment. Unlike better 19 studied models, mycobacteria express ~14% of their genes as leaderless transcripts. However, the 20 impacts of leaderless transcript structures on mRNA half-life and translation efficiency in 21 mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5' UTRs 22 to mRNA half-life and translation efficiency are similarly unknown. In both M. tuberculosis and 23 M. smegmatis, the essential sigma factor, SigA, is encoded by an unstable transcript with a 24 relatively short half-life. We hypothesized that sigA's long 5' UTR caused this instability. To test 25 this, we constructed fluorescence reporters and then measured protein abundance, mRNA 26 abundance, and mRNA half-life. From these data we also calculated relative transcription rates. 27We found that the sigA 5' UTR confers an increased transcription rate, a shorter mRNA half-life, 28 and a decreased translation rate compared to a synthetic 5' UTR commonly used in mycobacterial 29 expression plasmids. Leaderless transcripts produced less protein compared to any of the leadered 30 transcripts. However, translation rates were similar to those of transcripts with the sigA 5' UTR, 31 and the protein levels were instead explained by lower transcript abundance. A global comparison 32 of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in 33 protein:mRNA ratios for natural leadered and leaderless transcripts, consistent with the idea that 34 variability in translation efficiency among mycobacterial genes is largely driven by factors other 35 than leader status. The variability in mRNA half-life and predicted transcription rate among our 36 constructs could not be explained by their different translation efficiencies, indicating that other 37 factors are responsible for these properties and highlighting the myriad and complex roles played 38 by 5' UTRs and other sequences downstream of transcription start sites. 39 40 41 5' UTRs can also regulate gene expression by altering mRNA turnover rates. This can be a 57 consequence of altered translation rates, as impairments to translation often lead to faster mRNA 58 decay [16][17][18][19][20][21][22]. In other cases, mRNA stability is directly affected by sRNA binding to 5' UTRs or 59 by UTR secondary structure [9,[23][24][25][26][27][28]. In E. coli, the half-life of the short-lived transcript bla can 60 be significantly increased when its native 5' UTR is replaced with the 5' UTR of ompA, a long-61 lived transcript [29][30][31]. Conversely, deletion of ompA's native 5' UTR decreased its half-life by 62 5-fold [30]. The longevity conferred by the ompA 5' UTR was attributed to the presence of a non-63 specific stem-loop as well as the specific RBS sequence [30][31][32]. Secondary ...

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“…The A and B subunits of both Stx1 and Stx2 are encoded by separate genes, stxA and stxB, with stxB immediately downstream of stxA. Only 12 base pairs separate the A and B coding sequences, and there is no evidence for a separate promoter for the B subunit [5].…”

Section: Introductionmentioning

confidence: 98%

N-Terminus Leader Sequence of Shiga Toxin (Stx) 1 Is Essential for Production of Active Recombinant Protein in E. coli

Oloomi

Bouzari²,

Arshadi³

2006

PPL

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Escherichia coli clones expressing recombinant shiga toxin (Stx1) and its derivatives Stx1-A and Stx1-B subunits were established to release the proteins into periplasmic space. The expression was examined by SDS-PAGE to visualize the subunits. The secreted assembled subunits were extracted with polymyxin B and assessed for biological activity. The results showed that the presence of N-terminus leader sequence of the gene is essential for assembly of the subunits to yield biologically active holotoxin (AB5). The absence of the leader sequence did not affect the expression of the subunits but did disrupt the holotoxin assembly.

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Roles of a ribosome-binding site and mRNA secondary structure in differential expression of Shiga toxin genes

Cited by 17 publications

References 47 publications

The Role of a β Barrel Loop 4 Extension in Modulating the Physical and Functional Properties of Long‐Chain 2‐Hydroxy‐Acid Oxidase Isozymes

The Role of a β Barrel Loop 4 Extension in Modulating the Physical and Functional Properties of Long‐Chain 2‐Hydroxy‐Acid Oxidase Isozymes

The impact of leadered and leaderless gene structures on translation efficiency, transcript stability, and predicted transcription rates inMycobacterium smegmatis

N-Terminus Leader Sequence of Shiga Toxin (Stx) 1 Is Essential for Production of Active Recombinant Protein in E. coli

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