Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 S RNA and specific 50 S subunit proteins

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

“…They were eluted and ana- Smaller RNA fragment. In an earlier study (2), in which stronger digestion conditions were employed (30 min at 25°C instead of 10 min at 0°C), the predominant digestion product of the complexes lacked the stem region of the 5S RNA (nucleotides 1-11 and 111-120). The experiment was repeated for the L25-5S RNA complex in order to confirm the result and to establish whether protein was bound to this smaller fragment.…”

“…It cannot be concluded, therefore, as it was earlier (Gray et al, 1973), that this RNA fragment contains the primary binding site of L18.…”

“…The RNA was renatured by heating at 55°C for 10 min in HMK reconstitution buffer (30 mM Hepes-OH, 20 mM MgCl2, 300 mM KCl, pH 7.6) and slowly cooling (15). The presence of the native A-conformer was established electrophoretically (15) and by the specific binding of proteins L18 and L25 (2,16). More than 90% of the native conformer was invariably present.…”

“…L25 was also prepared by treating ribosomes at pH 3.8 as described by Brosius (19). Fraction 1 proteins, which correspond to a 2 M LiCl-split fraction from 50S subunits,,were prepared and identified, as described earlier (2,20 Oligonucleotide sequence analyses of the RNA fragments. The 32p-labelled fragments were located in slab gels autoradiographically and the gel pieces were excised.…”

“…Gray et al (1,2) first identified an RNA region which was "protected" by proteins L18 and L25 against ribonuclease A digestion. This region occurred within the sequences 1-11 and 69-120.…”