Figure 4

s194 and Hts4 RNAs interact with ribosomes. (A) Northern blot analysis of intergenic rancRNA candidates on RNA isolated from crude ribosome pellets (P100) or the corresponding supernatants (S100) of H. volcanii cells grown in normal medium to either exponential (exp) or stationary (stat) phase. (B) On the left a representative polysome profile of H. volcanii is shown. Fractions containing polysomes, 50S, or 30S subunits were analyzed on an agarose gel to confirm the rRNA identities. On the right northern blot signals for different rancRNA candidates in different gradient fractions are shown. H. volcanii cells were either grown to exponential (exp) or stationary (stat) phase. Full-length blots are presented in Supplementary Fig. 9. (C) In vitro filter binding studies of [³²P]-radiolabeled in vitro transcribed s194 RNA on purified ribosomal subunits (left panel, representative phosphorimager screen). As specificity control, an in vitro transcribed radiolabeled RNA with same length, but randomized nucleotide sequence (scr) was used. Right panel shows the mean and standard deviations of four experiments whereas binding of s194 RNA to 50S ribosomal subunit was set to 100%. (D) Increasing amounts of [³²P]-radiolabeled in vitro transcribed s194 RNA were added to 5 pmol purified H. volcanii 50S ribosomal subunits and binding efficiency was monitored by dot blot filter binding. The signals obtained in the absence of any ribosomal subunits (−50S) served as background and were subtracted from the corresponding samples containing 50S subunits (+50S). Lower panel shows the mean and standard deviation of three independent binding experiments.