Molecular Biology 5th Edition by Robert Weaver

Exercise 6

Consider the RNA sequencing results in Figure 14.42b. Knowing the cutting specificities of each enzyme, how do we know (a) that the band at the bottom in the first lane represents G (b) that the next band represents A (c) that the eighth band from the bottom represents C (d) that the 13th, 14th, and 15th bands from the bottom represent U's (Hint: PhyM cut inefficiently after U's in this experiment.)
Figure 14.42 Addition of GMP to the 5'-end of the excised intron. (a) Radioactive GTP labels the intron during splicing. Cech and coworkers transcribed plasmid pIVS11 under nonsplicing conditions with no labeled nucleotides. They isolated this unlabeled 26S rRNA precursor and incubated it under splicing conditions in the presence of [a- 32 P]GTP. Then they chromatographed the products on Sephadex G-50, electrophoresed the column fractions, and autoradiographed the gel. Lanes 1-4 are successive fractions from the Sephadex column. Lane 5 is a linear intron marker. Lanes 2 and 3 contain the bulk of the linear intron, and it is labeled, indicating that it had incorporated a labeled guanine nucleotide. (b) Sequence of the labeled intron. Cech and coworkers used an enzymatic method to sequence the 5'-end of the RNA. They cut it with base (OH - ), which cuts after every nucleotide; RNase Phy M, which cuts after A and U; RNase U2, which cuts after A; and RNase T1, which cuts after G. Treatment of each RNA sample is indicated at top. The deduced sequence is given at left. Note the 5'-G at bottom.