12.1 Nucleoside triphosphates labeled with [32P] at the a, b, or g position are useful for monitoring various aspects of transcription. For the specific process listed in a-c, give the position of the label that is appropriate for examining that step.
a) Initiation by E. coliRNA polymerase.
b) Forming the 5' end of eukaryotic mRNA.
c) Elongation by eukaryotic RNA polymerase II.
12.2 (POB) RNA posttranscriptional processing.
Predict the likely effects of a mutation in the sequence (5')AAUAAA in a eukaryotic mRNA transcript.
12.3 A phosphoester transfer mechanism (or transesterification) is observed frequently in splicing and other reactions involving RNA. Are the following statements about these mechanisms true or false?
a) The mechanism requires the cleavage of high-energy bonds from ATP.
b) The initiating nucleophile for splicing of Group I introns (including the intron of pre-rRNA from Tetrahymena) is the 3' hydroxyl of a guanine nucleotide.
c) The initiating nucleophile for splicing of nuclear pre-mRNA is the 2' hydroxyl of an internal adenine nucleotide.
d) The individual reactions in the phosphoester transfers are reversible, but the overall process is essentially irreversible because of circularization (includes lariat formation) of the excised intron.
12.4 What properties are shared by the splicing mechanism of Tetrahymena pre-rRNA and Group II fungal mitochrondrial introns?
12.5 Please answer these questions on splicing of precursors to mRNA.
a) What dinucleotides are almost invariably found at the 5’ and 3’ splice sites of introns?
b) Which splicing component binds at the 5' splice junction?
c) What nucleotides are joined by the branch structure in the intron during splicing?
d) What is ATP used for during splicing of precursors to mRNA?
12.6 (POB) RNA splicing.
What is the minimum number of transesterification reactions needed to splice an intron from an mRNA transcript? Why?
12.7 Match the following statements with the appropriate eukaryotic splicing process listed in parts a-c.
1) A guanine nucleoside or nucleotide initiates a concerted phosphotransfer reaction.
2) The consensus sequences at splice junctions are AG'GUAAGU...YYYAG'G (' is the junction, Y = any pyrimidine).
3) Splicing occurs in two separate steps, cutting to generate a 3'-phosphate followed by an ATP dependent ligation.
4) Splicing requires no protein factors.
5) Splicing requires U1 small nuclear ribonucleoprotein complexes.
a) Splicing of pre-mRNA.
b) Splicing of pre-tRNA in yeast
c) Splicing of pre-rRNA in Tetrahymena
12.8 The enzyme RNase H will cleave any RNA that is in a heteroduplex with DNA. Thus one can cleave a single-stranded RNA in any specific location by first annealing a short oligodeoxyribonucleotide that is complementary to that location and then treating with RNase H.
This approach is useful in determining the structure of splicing intermediates. Let's consider a hypothetical case shown in the figure below. After incubation of radiolabeled precursor RNA (exon1-intron-exon2) with a nuclear extract that is capable of carrying out splicing, the products were analyzed on a denaturing polyacrylamide gel. The results showed that the exons were joined as linear RNA, but the excised intron moved much slower than a linear RNA of the same size, indicative of some non-linear structure. The excised intron was annealed to a short oligodeoxyribonucleotide that is complementary to the region at the 5' splice site (labeled oligo 1 in the figure), treated with RNase H and analyzed on a denaturing polyacrylamide gel. The product ran as a linear RNA with the size of the excised intron (less the length of the RNase H cleavage site). As summarized in the figure, the excised intron was analyzed by annealing (separately) with three other oligodeoxyribonucleotides, followed by RNase H treatment and gel electrophoresis. Use of oligodeoxyribonucleotide number 2 generated a Y-shaped molecule, use of oligodeoxyribonucleotide number 3 generated a V-shaped molecule with one 5' end and 2 3' ends, and use of oligodeoxyribonucleotide number 4 generated a circle and a short linear RNA.
(a) What does the result with oligodeoxyribonucleotide 2 tell you?
(b) What does the result with oligodeoxyribonucleotide 4 tell you?
(c) What does the result with oligodeoxyribonucleotide 1 tell you?
(d) What does the result with oligodeoxyribonucleotide 3 tell you?
(e) What is the structure of the excised intron? Show the locations of the complementary oligos on your drawing.