K. Meisenheimer, P. Meisenheimer, Michael Willis
1996
Citations
0
Influential Citations
35
Citations
Journal
Nucleic acids research
Abstract
5-Iodouracil is an excellent chromophore for introduction intoboth RNA and DNA for the purpose of achieving high yieldphotocrosslinking of nucleoprotein complexes ( 1,2). Crosslinkingyields are often three to five times higher than those achieved with5-bromouracil, in part, because the 5-iodouracil chromophoreabsorbs at longer wavelength and consequently can be excitedmore selectively. The highest yields are achieved by irradiatingwith a helium cadmium laser emitting at 325 nm (1); however,useful yields can also be obtained with a 312 nm transilluminator(1,2). The technique is primarily selective for aromatic amino acidresidues; consequently, success in crosslinking depends uponproximity of a U or T of the nucleic acid to an aromatic ring of theprotein. We now report that 5-iodocytosine is an excellentchromophore for nucleoprotein photocrosslinking complementing5-iodouracil.The nucleoprotein complex selected for demonstrating photo-crosslinking with the 5-iodocytosine chromophore was an RNAhairpin within the genome of the bacteriophage MS2 bound to adimer of the phage coat protein. The system has been character-ized by a co-crystal structure ( 3), and photocrosslinking has beendefined with RNAs bearing 5-iodouridine (1) and 5-bromo-uridine ( 4,5) complexed with the almost identical R17 coat protein.Further, the effect of RNA sequence variations on protein bindingand crosslinking is well established (4). RNA 1 bearing a single5-iodouridine at the –5 position (Fig. 1) binds with high affinityto the coat protein and upon irradiation of the complex at 325 nm,crosslinks in 75–95% yield to Tyr85 of the coat protein (1,5).Comparable photocrosslinking is now demonstrated withRNA 2 (Fig. 1) bearing an iodocytidine at the –5 position and fiveadditional iodocytidines in stem positions. Irradiation at 325 nmgives 75–95% crosslinking after 2 h with only traces of RNAcleavage. A time course of the crosslinking is shown by theelectrophoretic gel in Figure 2. The high yield is dependent upona saturating protein concentration. With a substantial excess ofcoat protein but at a concentration sufficient to bind only 36% ofthe RNA throughout the irradiation, a maximum crosslinkingyield of only 20% is achieved even after 3 h of irradiation. Thebalance of the RNA appears in the electrophoretic gel (not shown)primarily as unreacted RNA (40%) and two RNA cleavageproducts (40%). Further, gel electrophoretic analysis of thereaction mixture from irradiation of the RNA in the absence ofcoat protein showed no high molecular weight bands and inparticular no bands at the location of the crosslinked nucleoprotein