The studies of cooperative regions in T7 RNA polymerase

Protasevich, I.I.; Memelova, L. V.; Kochetkov, S. N.; Makarov, Alexander А.

doi:10.1016/0014-5793(94)00718-7

Cited by 5 publications

(2 citation statements)

References 13 publications

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“…Two of them, S430P and N433T are located in the interface of NTD and CTD, S430P substitution enhances hydrophobic interaction with F268 and F416, thereby increase domain interaction (Figure 4B ). Scanning microcalorimetry measurement showed that the T m for CTD alone is 7°C lower than in the intact protein, strongly suggesting the CTD is stabilized by inter-domain interaction with the NTD ( 39 ). F880Y substitution generates hydrogen-bonding interactions with P474 and D879, thereby increasing internal stability of the CTD (Figure 4C ).…”

Section: Resultsmentioning

confidence: 99%

Transcription yield of fully 2′-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants

Meyer

Garry

Hall³

et al. 2015

Nucleic Acids Res

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On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2′-O-methyl modified RNA with yields much higher than enzymes currently employed.

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Section: Resultsmentioning

confidence: 99%

Transcription yield of fully 2′-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants

Meyer

Garry

Hall³

et al. 2015

Nucleic Acids Res

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“…Facile cleavage of the protein into 20‐kD N‐terminal and 80‐kD C‐terminal fragments (Davanloo et al 1984; Tabor and Richardson 1985) suggests the presence of at least two domains. Previous calorimetric studies (Protasevich et al 1994) showed that unfolding of T7RNAP proceeds in two stages under the conditions they used, corresponding to disruption of two structural regions in the molecule.…”

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confidence: 99%

Thermal and urea‐induced unfolding in T7 RNA polymerase: Calorimetry, circular dichroism and fluorescence study

et al. 2001

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Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased ␤-sheet and reduced ␣-helix content relative to the native state. Urea-induced unfolding at 25°C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15°to 60°C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25°C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

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Structural studies of arginine induced enhancement in the activity of T7 RNA polymerase

Pal

Das

Dasgupta

2012

Biochemical and Biophysical Research Communications

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The studies of cooperative regions in T7 RNA polymerase

Cited by 5 publications

References 13 publications

Transcription yield of fully 2′-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants

Transcription yield of fully 2′-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants

Thermal and urea‐induced unfolding in T7 RNA polymerase: Calorimetry, circular dichroism and fluorescence study

Structural studies of arginine induced enhancement in the activity of T7 RNA polymerase

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