Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H

Metelev, Valeri; Zayakina, G. V.; Ryabushenko, I.L.; Krynetskaya, N. F.; Романова, Е. А.; Oretskaya, T. S.; Shabarova, Zoe A.

doi:10.1016/0014-5793(88)81429-7

“…Previous experiments introducing pyrophosphate in the hybrid by Metelev et a!. (31) also support this deformation. Thus, the role of deoxyribonucleotide may be its flexibility to allow the hybrid to kink, so that interaction with the enzyme is enhanced.…”

Section: Discussionsupporting

confidence: 63%

How does RNase H recognize a DNA.RNA hybrid?

Nakamura

¹

,

Oda

²

,

Iwai

³

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA-RNA hybrid Escherichia coil RNase H complex. Sitedirected mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA-RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.

show abstract

“…There are reports that RNase H has a slight preference for the phosphodiester bonds adjacent to pyrimidines [28] and that a retroviral polypurine tract is resistant to retroviral RNase H degradation [29]. However, E. coli RNase HI also cleaves the phosphodiester bonds that are not adjacent to pyrimidines [15][16][17]221. Therefore, the preference of hybrids 0 and 1 to cleavage with E. coli RNase HI at A9-ClO and A19-C20 may not reflect the base preference of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of Escherichia Coli Ribonuclease HI Using Oligomeric DNA/RNA Substrates Suggests an Alternative Mechanism for the Interaction between the Enzyme and the Substrate

Kanaya

¹

,

Kanaya

²

1995

European Journal of Biochemistry

View full text Add to dashboard Cite

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-ClO. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.

show abstract

“…There are reports that RNase H has a slight preference for the phosphodiester bonds adjacent to pyrimidines [28] and that a retroviral polypurine tract is resistant to retroviral RNase H degradation [29]. However, E. coli RNase HI also cleaves the phosphodiester bonds that are not adjacent to pyrimidines [15][16][17]221. Therefore, the preference of hybrids 0 and 1 to cleavage with E. coli RNase HI at A9-ClO and A19-C20 may not reflect the base preference of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Analysis of Escherichia Coli Ribonuclease HI Using Oligomeric DNA/RNA Substrates Suggests an Alternative Mechanism for the Interaction between the Enzyme and the Substrate

Kanaya

¹

,

Kanaya

²

1995

Eur J Biochem

0

View full text Add to dashboard Cite

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-ClO. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.

show abstract

Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H

Abstract: Chimeric oligo(ribo-deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H. It has been shown that the use of d(TGTGTAT)ppGC-CAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C,, and A,, .

Cited by 16 publications

References 5 publications

How does RNase H recognize a DNA.RNA hybrid?

How does RNase H recognize a DNA.RNA hybrid?

Kinetic Analysis of Escherichia Coli Ribonuclease HI Using Oligomeric DNA/RNA Substrates Suggests an Alternative Mechanism for the Interaction between the Enzyme and the Substrate

Kinetic Analysis of Escherichia Coli Ribonuclease HI Using Oligomeric DNA/RNA Substrates Suggests an Alternative Mechanism for the Interaction between the Enzyme and the Substrate

Contact Info

Product

Resources

About