Scission of RNA by the chemical nuclease of 1,10-phenanthroline-eopper ion: preference for single-stranded loops

Murakawa, George J.; Chen, Chi‐hong B.; Kuwabara, Michio D.; Nierlich, Donald P.; Sigman, David S.

doi:10.1093/nar/17.13.5361

Cited by 69 publications

(55 citation statements)

References 17 publications

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“…Plasmid pGM820 contains the first 63 nucleotides of the lac operon message (26,27). After linearization of the plasmid with Hindlll, the probe was synthesized with SP6 RNA polymerase as described below.…”

Section: Methodsmentioning

confidence: 99%

Transcription and decay of the lac messenger: role of an intergenic terminator

et al. 1991

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Prior work has indicated that the polycistronic lacZYA mRNA of Escherichia coli is cleaved during decay at approximately intergenic sites (L. W. Lim and D. Kennell, J. Mol. Biol. 135: 369-390, 1979). In this work, we characterized the products by using probes specific for the different cistrons. This analysis indicated that six lac mRNA species are present in the following order of decreasing abundance: lacZ, -A, -ZYA, -ZY, -YA, and -Y. Very little lacYA and lacY mRNAs were present, whereas in cells induced to steady state, there was 10 times more lacZ than lacZYA mRNA. The lacZ mRNA appeared as a discrete species extending to a site in the lacZ-Y intergenic space (ca. residue 3150). This site is just distal to a potential rho-independent termination sequence. We examined the function of this sequence to determine whether it contributes to the distribution of the mRNAs. Although the termination sequence was shown to function in vitro, when it was recloned into an expression vector, no termination was seen in vivo. Moreover, direct examination of the kinetics of lac messenger synthesis revealed that after initiation, most transcription continued to the end of the operon. We conclude that during normal growth, the operon is transcribed in its entirety and that the individual lac mRNAs are formed by cleavage. These results confirm earlier work implying that the lac operon is transcribed in its entirety but are in conflict with several recent reports suggesting that internal termination occurs. Our findings indicate that the natural polarity of the operon (lacZ is expressed sixfold more strongly than lacA) is based on posttranslational effects and not on polarity of transcription.There have been extensive studies of the transcription and decay of the lac operon mRNA (reviewed in reference 18). According to current understanding, the lacZYA mRNA is transcribed polycistronically and then is cleaved at approximately intergenic sites. These cleavages, near the 5' ends of the lacY and lacA transcripts and at an additional site near the 5' end of the lacZ transcript, inactivate the distal mRNAs. The further chemical decay of these mRNAs then occurs as a net 5'-to-3' process. In this process, endonucleolytic cleavages occur on the mRNA that is exposed as ribosomes run off inactivated messages. The resulting fragments are then removed by the 3'-to-5' exonucleases, RNase II, polynucleotide phosphorylase and possibly other enzymes (1,14,18). The endonucleolytic cleavage sites on the lacZ and lacY species have been characterized but the enzyme(s) responsible for the endonucleolytic cleavages has not been identified (7,34). It appears, however, that RNase III is not involved in lac mRNA decay, although it does inactivate a few other mRNA species (2, 18, 27a). A newly described endonuclease has some properties inferred for the enzyme that cleaves the lac transcripts (6).The cleavage of the lacZYA mRNA was first deduced from the observation that the lacA mRNA decays about twice as fast as lacZ mRNA (19). Subsequently, the cleavag...

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Section: Methodsmentioning

confidence: 99%

Transcription and decay of the lac messenger: role of an intergenic terminator

et al. 1991

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“…Cu-(phen) 2 is a chemical nuclease with specific three-dimensional structure that has been previously shown to recognize, i.e. bind and cleave, RNA helix distortions (50). The reactivity of TfR-IREs in the context of the 5xRNA, annealed the same way as for the IRP binding and for the nuclease resistance experiments, was markedly changed compared with single TfR-IREc (32).…”

Section: Detected a Specific Structure Of Tfr-ires In The Context Of mentioning

confidence: 99%

“…A structural basis for the observed discrepancy was provided by probing the RNA with a shape-selective transition metal complex, Cu-(phen) 2 , a 1:2 complex of Cu and 1,10-phenanthroline that binds to small bends and loops of tRNA. Redox properties of Cu-(phen) 2 yield radicals that cut RNA in the vicinity of the RNA bending site (50). Cu-(phen) 2 specifically binds the internal loop/bulge of the ferritin IRE, but appears to be too large to bind the C-bulge in other IREs (3,50) or in ferritin IRE with U 6 deletion in the IL/B, that converts the IL/B in the helix to a bulge (32,33).…”

Section: Mtementioning

confidence: 99%

“…Redox properties of Cu-(phen) 2 yield radicals that cut RNA in the vicinity of the RNA bending site (50). Cu-(phen) 2 specifically binds the internal loop/bulge of the ferritin IRE, but appears to be too large to bind the C-bulge in other IREs (3,50) or in ferritin IRE with U 6 deletion in the IL/B, that converts the IL/B in the helix to a bulge (32,33). The site of Cu-(phen) 2 binding in the helix of the ferritin mRNA is the same site associated with metal binding, proton-induced disorder, and IRP2 binding in solution (17,32).…”

Section: Mtementioning

confidence: 99%

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Multiple, Conserved Iron-responsive Elements in the 3′-Untranslated Region of Transferrin Receptor mRNA Enhance Binding of Iron Regulatory Protein 2

Erlitzki

Long

Theil

2002

Journal of Biological Chemistry

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Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3-untranslated region (TfR-3-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen) 2 binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.

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“…Primer extension assays were used to quantify each of the insulin mRNAs as described (24)(25)(26). Each oligonucleotide (oligo) is listed in Table I and the corresponding region of the insulin gene depicted in Fig.…”

Section: Methodsmentioning

confidence: 99%

Adaptation to supraphysiologic levels of insulin gene expression in transgenic mice: evidence for the importance of posttranscriptional regulation.

Schnetzler

Murakawa²,

Abalos³

et al. 1993

J. Clin. Invest.

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Insulin production was studied in transgenic mice expressing the human insulin gene under the control of its own promoter. Glucose homeostasis during a 48-h fast was similar in control and transgenic mice, with comparable levels of serum immunoreactive insulin. Northern blot and primer extension analyses indicated that more than twice as much insulin mRNA is present in pancreata from transgenic mice. Primer extension analysis using oligonucleotides specific for mouse insulins I and II or for human insulin, showed that the excess insulin mRNA was due solely to expression of the foreign, human insulin gene. The ratio of mRNA for mouse insulin I and II was unaffected by coexpression of human insulin. There were coordinate changes in the levels of all three mRNA during the 48-h fast, or after a 24-h fast followed by 24-h refeed. Despite the supraphysiologic levels of insulin mRNA in the transgenic mice, their pancreatic content of immunoreactive insulin was not significantly different from controls. The comparison of the relative levels of human and mouse insulin mRNAs with their peptide counterparts (separated by HPLC) indicates that the efficiency of insulin production from mouse insulin mRNA is greater than that from human, stressing the importance of posttranscriptional regulatory events in the overall maintenance of pancreatic insulin content. (J. Clin. Invest. 1993. 92:272-280.)

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Scission of RNA by the chemical nuclease of 1,10-phenanthroline-eopper ion: preference for single-stranded loops

Cited by 69 publications

References 17 publications

Transcription and decay of the lac messenger: role of an intergenic terminator

Transcription and decay of the lac messenger: role of an intergenic terminator

Multiple, Conserved Iron-responsive Elements in the 3′-Untranslated Region of Transferrin Receptor mRNA Enhance Binding of Iron Regulatory Protein 2

Adaptation to supraphysiologic levels of insulin gene expression in transgenic mice: evidence for the importance of posttranscriptional regulation.

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