[10] Preparation of 5S RNA-related materials for nuclear magnetic resonance and crystallography studies

Moore, Peter B.; Abo, S. R.; Freeborn, Betty; Gewirth, D.T.; Leontis, Neocles B.; Sun, Gongqin

doi:10.1016/s0076-6879(88)64041-9

Cited by 20 publications

(21 citation statements)

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“…The preparation of the RNAs discussed below have been described elsewhere: (1) for 5S RNA and 5S RNA fragment 1, see (11), (2) for pDG07 RNA, see (7), and (3) for the G102A and U74C mutants of pDG07 RNA, see (8). pw39 RNA is a T7 RNA polymerase transcript made using a synthetic DNA template (13,14,10).…”

Section: Materials and Methods Nmr Samplesmentioning

confidence: 99%

A study of the conformation of 5S RNA by³¹P NMR

Zhang¹,

Rycyna²,

Moore³

1989

Nucl Acids Res

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Only a small number of resolved, single phosphorous, phosphodiester resonances are observed in the 31p spectrum of the 5S rRNA from E. coli. Its spectrum is much simpler than that of a tRNA (Gueron, M. and Shulman, R.G. (1975) Proc. Natl. Acad. Sci. 72, 3482-3484), which suggests that 5S RNA does not have a tightly folded, tRNA-like, tertiary structure. The resolved resonances in the 5S spectrum originate in loops D and E, near bases 88 and 76, respectively.

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Section: Materials and Methods Nmr Samplesmentioning

confidence: 99%

A study of the conformation of 5S RNA by³¹P NMR

Zhang¹,

Rycyna²,

Moore³

1989

Nucl Acids Res

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“…For example, RNA transcripts overexpressed in vivo using the T7 system 9 are long and heterogeneous, as a consequence of terminator read-through, and have never been 2 purified. 5S ribosomal RNA 10 and tRNA [11][12][13][14] are two exceptions which have been successfully expressed in E. coli. In the latter case, it works because tRNA are recognized by cellular enzymes that precisely process the primary transcript, incorporate modified nucleotides and repair their 3'-end 15 .…”

Section: Introductionmentioning

confidence: 99%

Recombinant RNA technology: the tRNA scaffold

2007

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During the past decades, RNA has emerged as a major player in most cellular processes.Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. So far, this has been a bottleneck, as the only methods for producing such pure RNA were in vitro syntheses.Here, we describe a generic approach for expressing and purifying structured RNA in E.coli, using a series of tools which parallel those available for recombinant proteins. Our system is based on a camouflage strategy, the "tRNA scaffold", in which the recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular ribonucleases. This opens the way to large scale structural and molecular investigations of RNA function. IntroductionSystematic studies of protein structure, function and interactions such as structural genomics and double-hybrid approaches have greatly benefited from the development of efficient recombinant expression systems. Simultaneously, new roles have been evidenced for structured RNA in translation, chromosome maintenance, viral replication, as riboswitches or ribozymes [1][2][3] . As a result, these RNA are considered as promising drug targets, as for instance bacterial rRNA (antibiotics) or telomerase RNA 4 . Owing to their large structural repertoire, folded RNA are also interesting molecules for constructing self-assembling nano-objects, some of which have been used for delivering therapeutic siRNA 5,6 . As opposed to proteins, systematic biochemical, structural and pharmacological studies of RNA have however been hampered by difficulties in obtaining homogeneous samples in large quantities. So far, RNA has mostly been produced in vitro using either T7 RNA polymerase transcription 7 or chemical synthesis 8 , which are costly and cumbersome. Producing recombinant RNA in vivo could be an alternative, but thus far has been plagued by a number of obstacles, such as heterogeneity of the products, degradation by ribonucleases and difficulty in purifying the RNA from cell extracts. For example, RNA transcripts overexpressed in vivo using the T7 system 9 are long and heterogeneous, as a consequence of terminator read-through, and have never been 2 purified. 5S ribosomal RNA 10 and tRNA [11][12][13][14] are two exceptions which have been successfully expressed in E. coli. In the latter case, it works because tRNA are recognized by cellular enzymes that precisely process the primary transcript, incorporate modified nucleotides and repair their 3'-end 15 . This results in a single product of defined length, a key feature for structural studies. Furthermore, tRNA adopt a compact 3D structure which makes them resistant to both unfolding and nucleases.We used this robustness and precise processing to express other structured RNA in vivo and designed a generic method using tRNA as a protective scaffold. We extracted the desired recombinant RNA with high yield and purified it to homogeneity using standard chromatography. We employ tools ...

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“…The new data enable us to complete the assignment of the imino proton spectrum of fragment 1 we began several years ago (Kime & Moore, 1983b). Spectroscopic results on several mutant RNAs The method used for preparing ribosomal protein L25 may be found elsewhere (Moore et al, 1988). The 15N-labeled fragment 1 molecules used here were only partially labeled.…”

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An NMR study of the helix V-loop E region of the 5S RNA from Escherichia coli

Zhang

Moore²

1989

Biochemistry

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Experiments are described that complete the assignment of the imino proton NMR spectrum of the fragment 1 domain from the 5S RNA of Escherichia coli. Most of the new assignments fall in the helix V-loop E portion of the molecule (bases 70-78 and 98-106), the region most sensitive to the binding of ribosomal protein L25. The spectroscopic data are incompatible with the standard, phylogenetically derived model for 5S RNA, which makes all the base pairs possible in loop E with the sequences aligned in parallel (C70-G106, C71-G105, etc.) [see Delihas et al. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190]. Furthermore, the alternative loop E model proposed for spinach chloroplast 5S RNA by Romby et al. [(1988) Biochemistry 27, 4721-4730] does not apply to the closely homologous 5S RNA from E. coli. The 5S RNAs from E. coli and spinach chloroplasts do not have the same secondary structures in solution despite their strong sequence homologies, and neither appears to conform to the standard model for 5S RNA in the loop E region.

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[10] Preparation of 5S RNA-related materials for nuclear magnetic resonance and crystallography studies

Cited by 20 publications

References 10 publications

A study of the conformation of 5S RNA by³¹P NMR

A study of the conformation of 5S RNA by³¹P NMR

Recombinant RNA technology: the tRNA scaffold

An NMR study of the helix V-loop E region of the 5S RNA from Escherichia coli

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[10] Preparation of 5S RNA-related materials for nuclear magnetic resonance and crystallography studies

Cited by 20 publications

References 10 publications

A study of the conformation of 5S RNA by31P NMR

A study of the conformation of 5S RNA by31P NMR

Recombinant RNA technology: the tRNA scaffold

An NMR study of the helix V-loop E region of the 5S RNA from Escherichia coli

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A study of the conformation of 5S RNA by³¹P NMR

A study of the conformation of 5S RNA by³¹P NMR