The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo

Zhang, Shaoping; Stancek, Martin; Isaksson, Leif A.

doi:10.1093/nar/25.21.4301

Cited by 3 publications

(3 citation statements)

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“…Recall that the self-cleavage of a ribozyme was measured as 45% in E. coli versus 70±79% in vitro. In this extract, we find that ribozyme self-cleavage becomes almost undetectable, based on labelling of newly synthesized RNA with [ 32 P]UTP, and examination of products on a denaturing gel [40]. By contrast, long RNA hairpin loops seem to reduce protein synthesis strongly, by a translational effect rather than by some reduced stability of the RNA (as seen in gels).…”

Section: Strong Influence Of Rna Hairpin Loops On Protein Synthesis Imentioning

confidence: 84%

RNA hairpin loops repress protein synthesis more strongly than hammerhead ribozymes

Drew

Lewy

Conaty

et al. 1999

European Journal of Biochemistry

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A general study has been carried out to determine how well hammerhead ribozymes might reduce levels of specific protein synthesis in living cells, compared with RNA hairpin loops as stable but noncleaving controls. Four different experiments are described. First, a wide variety of hammerhead ribozymes, as well as hairpin loops, was cloned into a gene-expression cassette for b-galactosidase, upstream of the coding sequences for that reporter gene, and expressed from plasmids in several strains of Escherichia coli. The results show that ribozymes, when acting intramolecularly in E. coli, do not significantly reduce the amount of protein synthesized from any construct. As a control, long RNA hairpin loops do greatly reduce the amount of protein made. Secondly, we studied the transcription±translation of these same plasmids in a cell extract from E. coli. Once again, hammerhead ribozymes show no effect on levels of b-galactosidase, whereas long RNA hairpin loops produce a strong reduction, by apparent attentuation at the level of translation. Thirdly, we added an SV40 promoter to each plasmid, in order to study the effects of these gene-regulators on protein synthesis in Chinese hamster ovary cells. Here active intramolecular ribozymes produce a slight reduction in b-galactosidase, whereas long RNA hairpin loops produce an even stronger reduction than before. Those hairpin loops apparently induce degradation of their own mRNA in Chinese hamster ovary cells, by a mechanism not seen in E. coli. Finally, analyses of total RNA by S1-trimming show that hammerhead ribozymes will self-cleave a mRNA by a total of no more than 45±50% in E. coli, compared with 70±80% in vitro. Other analyses using Northern blotting were unable to detect any ribozyme cleavage in E. coli or Chinese hamster ovary cells. In summary, the ability of hammerhead ribozymes to reduce protein synthesis appears weak or nonexistent in all the cellular systems tested. By comparison, long RNA hairpin loops reduce protein synthesis strongly: by an apparent attentuation mechanism in E. coli or by a novel degradation of their own mRNA in Chinese hamster ovary cells.

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Section: Strong Influence Of Rna Hairpin Loops On Protein Synthesis Imentioning

confidence: 84%

RNA hairpin loops repress protein synthesis more strongly than hammerhead ribozymes

Drew

Lewy

Conaty

et al. 1999

European Journal of Biochemistry

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“…1 ) and pM9EGFP-hDHFR, respectively. Active (RzI) and inactive (RzII) hammerhead ribozyme sequences previously tested with reporter genes in E. coli [ 52 ] were obtained by PCR using oligos RzI_F + RzI_R and RzII_F + RzI_R (Table S 1 ), respectively. The RzI sequence is a variant of the natural hammerhead sequence from the satellite RNA of tobacco ringspot virus [ 53 , 54 ].…”

Section: Methodsmentioning

confidence: 99%

A cell-based ribozyme reporter system employing a chromosomally-integrated 5′ exonuclease gene

Aroonsri

Kongsee

Gunawan

et al. 2021

BMC Mol and Cell Biol

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Background Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. Results We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell. Conclusions The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.

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“…The ribozyme has been coupled to an aptamer to cleave off the part of the 5'-UTR sequestering the RBS only when a ligand is bound. (33,34). A similar synthetic construct has been applied in the 3'-UTR of eukaryotic transcripts, where the ribozyme cleaves off the poly-A tail upon binding by a ligand.…”

Section: Introductionmentioning

confidence: 99%

Structure-function relations of RNA molecules involved in gene expression and host defence

Creutzburg¹

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General introduction and thesis outline of growth. By that time, all exponential growth had stopped already and carry-over thymidine was consumed, preventing further growth. The bacteria cultivated in Greiner tubes, however, did not suffer from evaporation, so a very small subpopulation having a slightly increased thyA expression may become dominant overnight. It indicates that the background expression of ThyA is only just below the minimal requirement sometimes exceeding it. This very precise tuning weakens the robustness of the system as a biosensor.

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The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo

Cited by 3 publications

References 37 publications

RNA hairpin loops repress protein synthesis more strongly than hammerhead ribozymes

RNA hairpin loops repress protein synthesis more strongly than hammerhead ribozymes

A cell-based ribozyme reporter system employing a chromosomally-integrated 5′ exonuclease gene

Structure-function relations of RNA molecules involved in gene expression and host defence

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