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Text: To further narrow the rRNA region and the number of proteins implicated in SrmB binding , the purified SrmB - TAP complex was mildly digested with RNase A. This treatment should remove all rRNA regions that are not protected by SrmB , and all proteins that bind to these regions but do not interact directly with SrmB. The digested product was then re - purified on the calmodulin column ( SrmB retains the CBP after TAP purification ) , and analyzed for its RNA and protein content . Whatever the RNase A concentration in the range 0.05 - 0.5 mg / ml , the $ 0.5 - kb RNA fragment was converted into a $ 0.2 - kb fragment ( Figure 2A ) . After purification by urea - PAGE , this fragment was probed on a northern blot with oligonucleotide probes complementary to different regions of domain I ( Figure 2D ) . An in vitro transcript encompassing domain I was run in parallel as a control . Whereas the $ 0.2 - kb species hybridized with probes b ( 192 - 214 nt ) , c ( 240 - 259 nt ) and d ( 312 - 331 nt ) , no signal was detected with probes a ( 128 - 147 nt ) and e ( 409 - 428 nt ) ( Figure 2B ) . Thus , the $ 0.2 - kb product corresponds to the central region of domain I. Using oligonucleotide c for primer extension , its 5 0 extremity was mapped to nucleotide 198 ( data not shown ) . Therefore , the fragment spans nt 198 to $ 400 of 23S rRNA . To further confirm this localization with a different technique , the $ 0.2 - kb product was labeled at its 5 0 -end and partially digested with RNase T1 , as above . The profile of the digest unambiguously matched the succession of Gs in the central region of domain I. This is illustrated on Figure 2C , where a part of the gel is aligned with the 23S sequence from G263 to G291 . The protein composition of the partially digested SrmB complex showed that , compared with the undigested control , the number and amount of minor proteins were greatly reduced , resulting in a complex containing essentially SrmB , L4 and L24 ( compare lanes ' À'and ' + ' on Figure 2E ) . In particular , band 2 was no longer detected . Mass spectrometry confirmed the identity of the r - proteins . Of note , the binding sites for L4 and L24 both lie within the 0.2 - kb RNA fragment [ ( 16 ) ; Figure 2D ] , and they are in close proximity in the 3D - structure of the ribosome ( Figure 2F ) .<|eot_id|><|start_header_id|>assistant<|end_header_id|>

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