Basepairing potential of the 3′ terminus of 16S RNA: dependence on the functional state of the 30S subunit and the presence of protein S21

Abstract

The deoxyoctanucleotide 5′d(AAGGAGGT) which is complementary to the 3′ terminus of 16S RNA has been used as a probe to measure the potential of this rRNA region to engage in intermolecular basepairing. The site specific binding of the octanucleotide is shown by labeling 16S RNA in situ at its 3′ end with [³²P]pCp and T4 RNA ligase (EC 6.5.1.3.). The label can be released as pA[³²P]pCp by the simultaneous action of RNAse H (EC 3.1.4.34) and 5′d(AAGGAGGT). We show that (1) 30S subunits prepared according to standard procedures, bind less than one copy of 5′d(AAGGAGGT); (2) isolated 16S RNA and 30S subunits inactivated by transient exposure to 0.5 mM Mg²⁺ do not bind the octanucleotide; (3) binding to inactive subunits can be restored by a brief heat treatment; (4) 30S subunits lacking protein S21 do not bind 5′d(AAGGAGGT) even when submitted to heat treatment; (5) addition of protein S21 to subunits lacking S21 restores octamer binding; (6) the apparent exposure of the 16S RNA 3′ terminus brought about by protein S21 is accompanied by the potential of the subunits to accept MS2 RNA as messenger; (7) the presence or absence of S1 on 30S subunits has no effect on their octanucleotide binding property.