Six restriction enzymes discussed in Sequence-specific cleavage of RNA by Type II restriction enzymes (Murray et al.) have the ability to detect and cut RNA strands with that enzyme's recognition sequence.

As you know, restriction enzymes come from a system carried by some bacteria referred to as a restriction-modification system.

With this fact in mind while reading the paper above, I wondered how the bacteria carrying one of these six systems protects it's own RNA from restriction. Do the corresponding methyltranferase in each system methylate DNA as well as RNA? Would this not greatly sacrifice gene expression rates seeing as the methyltranferase surely cannot methylate all the RNA sequences being produced?

Perhaps I misunderstood the paper somehow. Could it be only in some circumstances outside the cell that these restriction enzymes have this unique ability?

  • $\begingroup$ When you take a look at Table 2 in the article, the activity to cleave RNA–DNA substrates is quite low. This means you should be careful to think that the enzymes actually cut RNA–DNA substrate in bacteria. The activity could be just artificial and may not happen physiologically. $\endgroup$ – 243 Jun 10 '15 at 22:40

This paper found that these enzymes recognize RNA:DNA heteroduplexes. Such duplexes are unlikely to be encountered in vivo. They are present when DNA is primed for replication, but these duplexes are relatively short and thus are less likely to randomly contain a recognition sequence. Furthermore, if the recognition sequence is found in the primer, the gDNA will be methylated specifically to protect it from degradation. RNA:DNA duplexes are also found during transcription, but they are also short and the gDNA would be methylated. Additionally, the transcription bubble where the heteroduplex forms is protected by RNA polymerase, which subsequently displaces the transcript from the gDNA as it leaves the elongating complex.

It's also important to note that this paper found that these enzymes don't cleave RNA:DNA as well as they do DNA:DNA duplexes and these results occurred under experimental conditions with relatively high concentrations of enzyme and substrate. The lower catalytic efficiency is likely due to lower binding affinity, which could be caused by:

  • presence of uracil in RNA (this was explicitly mentioned in the paper)
  • 2'-OH presenting a steric barrier
  • helix shape (RNA tends to adopt an A-form helix)
  • $\begingroup$ Further into the paper they discuss the ability of these enzymes (specifically TaqI and HaeIII I believe) to also cleave RNA alone. $\endgroup$ – CDB May 11 '15 at 17:27
  • $\begingroup$ @CDB Duplex RNA? I shall read the paper later and delete my answer if it's incorrect. $\endgroup$ – canadianer May 11 '15 at 17:29
  • $\begingroup$ Again, I could very easily be wrong, but that is my understanding of the paper. Feel free to correct me. $\endgroup$ – CDB May 11 '15 at 17:33
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    $\begingroup$ @CDB I don't know that they can't cleave dsRNA, it's just that this paper doesn't talk about it. I would expect that the 2'-OH presents a steric barrier since these enzymes evolved to recognize dsDNA. It seems from this paper that RNA:DNA duplexes lower the binding interaction so that, with RNA:RNA duplexes, the interaction may not be strong enough for efficient catalysis. $\endgroup$ – canadianer May 11 '15 at 19:43
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    $\begingroup$ @CDB The shape of the helix may play a role as well, RNA double helices tend to favor the A form of the helix, while DNA double helices tend to favor the B form. The A form might be just different enough that the binding sites don't like it. $\endgroup$ – user137 May 11 '15 at 21:01

As to why they don't recognize RNA-DNA heteroduplexes (which are present during transcription, for example), I suspect that the methylation which protects bacterial genomic dsDNA (see the DNA modifying enzyme section of this Columbia University lecture for more info) also protects RNA-DNA hybrids, as the genomic DNA would still be methylated.

Note that most of the enzymes featured in the paper are listed by New England Biolabs as methylation sensitive (for example: AvaII, BanI, HinfI, TaqI).

@canadianer and @user137 provide a host of reasons that these restriction enzymes cannot cleave ds/ssRNA in the comments on canadianer's answer (different helix form, different interaction strengths between strands).

  • $\begingroup$ RNA-DNA heteroduplexes (which are present during transcription) are transient and are unlikely to be targeted by either RNAse-H or these restriction enzymes. $\endgroup$ – WYSIWYG Jun 10 '15 at 19:03
  • $\begingroup$ True, but it also doesn't take very long for a restriction endonuclease to cleave dsDNA $\endgroup$ – Luigi Jun 10 '15 at 19:04
  • $\begingroup$ I just modified my comment. If such heteroduplexes really formed then the RNA will be first degraded by RNAse-H which is abundant. That, however does not happen $\endgroup$ – WYSIWYG Jun 10 '15 at 19:05
  • $\begingroup$ Yes, that was basically canadianer's answer. I wanted to provide additional insight based on bacterial self-methylation of its restriction enzyme recognition sites $\endgroup$ – Luigi Jun 10 '15 at 19:17
  • $\begingroup$ @WYSIWYG Maybe this justification is clearer. Q: Why don't bacterial restriction enzymes degrade RNA? canadianer: That paper talks about RNA-DNA heteroduplexes, which are rare in vivo. me: Also, a RNA-DNA heteroduplex would likely not be recognized for the same reason that bacterial genomic DNA isn't recognized. $\endgroup$ – Luigi Jun 10 '15 at 19:22

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