Functional interaction between RNase III and the Escherichia coli ribosome

Abstract

Background: RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria. Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity.

Results: We have analyzed RNase III cleavage sites around both ends of pre-23 S rRNA in the ribosome and in the protein-free pre-rRNA. It was found that in the protein-free pre-23 S rRNA the main cleavage site is at position (-7) in respect of the mature 5' end. When pre-23 S rRNA was in 70 S ribosomes or in 50 S subunits, the RNase III cleavage occurred at position (-3). We have demonstrated that RNase III interacts with both ribosomal subunits and with even higher affinity with 70 S ribosomes. Association of RNase III with 70 S ribosomes cannot be dissociated by poly(U) RNA indicating that the binding is specific.

Conclusions: In addition to the primary and secondary structural elements in RNA, protein binding to substrate RNA can be a determinant of the RNase III cleavage site.

Figure 1

RNase III cleavage sites in the 5' strand of pre-23 S rRNA. 70 S ribosomes and 50 S subunits were isolated from the RNase III positive (XL-1B) and from RNase III negative (CAG) strain. The 30 S rRNA precursor was transcribed in vitro. Samples were treated with RNase III (+ lanes). Control samples were incubated without the enzyme. RNase III cleavage sites on the 23 S rRNA in the ribosomes or in the naked RNA were determined by a reverse transcriptase primer extension. The sequence of the rrnB operon around the 5' end of 23 S rRNA is shown on lines A, C, G, T. RNase III cleavage sites and the mature 5' end of 23 S rRNA are indicated on the right (-7, -3, and 5' 23S).

Figure 2

RNase III cleavage sites in the 3' strand of pre-23 S rRNA. 70 S ribosomes, 50 S subunits or in vitro transcribed 30 S pre-rRNA were incubated with or without RNase III (+ and - lanes respectively). The cleavage sites were determined by S1 nuclease mapping. Sequencing lanes A, C, G, and T were used to determine of exact length of protected 3' [³²P]DNA. The positions of the RNase III cleavage and the mature 3' end of 23 S rRNA are indicated on the right.

Figure 3

Secondary structure of the end region of pre-23 S rRNA (processing stem). Mature 23 S rRNA is shown in bold. The 5' and 3' ends and the RNase III cleavage sites on the naked pre-23 S rRNA are indicated by solid lines and the cleavage sites in the ribosomes by dotted lines.