The splicing of a group I intron is studied in vitro. Lane 1 shows the pattern o
ID: 143894 • Letter: T
Question
The splicing of a group I intron is studied in vitro. Lane 1 shows the pattern observed from a radiolabled 350 nucleotides-long precursor, which contains a 200 nucleotide long intron, a 50 nucleotide long exon1 and a 100 nucleotide long exon2. After reaction the RNAs are detetected by autoradiography Draw the pattern of bands expected in the following conditions (all components required for the reaction have been added): Lane 2:-assume that each step of the reaction is not 100% efficient Lane 3-assume that each step in the reaction is 100% efficient (For all subsequent conditions, assume each step is 100% efficient) Lane 4 -Mutation of the wG in the intron to a C Lane 5 - Addition of a large excess of CI Lane 6 - The RNA is not radiolabeled but incubated with radiolabeled ATP Lane 7 - The RNA is not radiolabeled but incubated with radiolabeled GMP. 400 nt 300 nt 250 nt 200 nt 150 nt 100 nt 50 nt 2 3 4 7Explanation / Answer
Total size of RNA = 350 nucleotides
Size of intron (present between exon 1 and exon 2) = 200 nucleotides
Size of exon 1 = 50 nucleotides
Size of exon 2 = 100 nucleotides
Lane 1 : total RNA present, 350 nt long
Lane 2: as the steps of intron splicing are not 100% efficient, we will get different length of RNAs relative to each step. A thin band of 350 nt is seen because no step has occurred in some RNAs. A medium band of 300nt is got as it is aberrantly spliced from intron and exon 1 joint and exon 2 and intron makes this band ( 100+ 200). A medium band of 200nt is seen. This is of only intron. So eventually in some RNA, after all steps introns are cut out. A medium band of 150 nt suggests that two exons are joined (exon 1 + exon 2= 100 + 50). A medium band of 50 suggests that only exon 1 is present and due to incorrect steps it is not joined with exon 2.
Lane 3: As all steps are 100% efficient we will get perfectly spliced RNA with two exons joined to form 150nt band. A 200nt band is there to indicate removed intron from RNAs.
Lane 4: As G is present in intron and exon 2 junction as splice site and it is mutated to C, splicing between intron and exon 2 will not occur. But splicing between intron and exon 1 will take place and hence we got 2 bands. One of exon 1 which is 50nt long and one of joined intron plus exon 2 (200+100) which is 300nt long.
Lane 5: in excess Cl- splicing reactions will stop so we get only one band of RNA which totally unspliced and of 350 nt long.
Lane 6. No band will come as in RNA uracil is incorporated as base instead of adenine. So radiolabelled ATP will not be incorporated in RNA and no band will come in autoradiography.
Lane 7: it is radiolabelled GMP which was used to incubate. In group I intron splicing GMP is required at splicing junction. So only intron and exon 2 joint is seen which is of 200+100=300nt long.
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