Inactivation of the polymerase would lead to incomplete pre-mRNA processing, including 3′ end formation, splicing, editing, and transport—and would certainly be lethal.

Two; one to cleave the 5′ exon-intron junction and one to join the two exons, with release of the intron.

Group I and group II introns are generally self-splicing, with only the RNA backbone of the intron required for the reaction in vitro. The nucleophile in the first step (cleavage of the 5′ splice site) for group I introns is a guanine nucleotide or nucleoside, and for group II introns is the 2′-OH of an internal A residue. The second step uses the liberated 3′-OH at the 5′ splice site as a nucleophile to attack the phosphodiester bond at the 3′ splice site, joining the exons. Site specificity is aided by guide sequences that are part of the intron structures. Introns removed with spliceosomes follow a mechanism similar to that for group II introns, but also rely on the spliceosome ribonucleoprotein complex to catalyze their excision.

Self-splicing catalyzes phosphodiester exchange reactions with no net loss or gain of energy. Bonds are broken and re-formed with different nucleotides, and there is no change in the number of phosphodiester bonds, just in the covalent bonding partners. Thus, there is no net change in free energy from reactants to products.

Incubate the total RNA in the presence of an appropriate buffer to ensure RNA folding. Add a fluorescently labeled guanine nucleotide analog to label the 5′ ends of any RNA that uses this nucleotide as a nucleophile in the first step of splicing. After reaction, fractionate the RNA (by size) on a polyacrylamide gel and look for fluorescently labeled RNAs. Controls could include incubation with other labeled nucleotides or with just the fluorescent dye, in parallel reactions—which are not expected to result in labeled RNAs, based on the group I intron reaction mechanism.

S-16

Over many generations, the wild-type bacteria would win out and the mutant strains would disappear. The normal rRNA modifications add subtle but important thermodynamic stability to the ribosome structure, enabling more robust protein synthesis in the wild-type bacteria.

Sometimes. By definition, enzymes remain unchanged after catalysis, which is not the case for self-splicing and self-cleaving RNAs. They are catalysts because they enhance the rate of bond cleavage and/or joining, but in their natural form they have only one reaction turnover. However, some of these RNAs can be engineered to bind to separate substrate molecules and catalyze reactions on multiple such molecules, so they are inherently capable of functioning as enzymes. Some, such as RNase P, do so in their natural state.

The binding and hydrolysis of GTP by Ran ensures a cycle in which mRNA is bound in the nucleus and released in the cytoplasm, and not the reverse.

The lifetime would increase, because mRNAs in higher eukaryotes are degraded by 5′→3′ exonucleolytic digestion.

The most commonly altered bases are cytosine and adenine. The alterations generally involve deamination reactions that convert these bases to uracil and inosine, respectively. Uracil pairs with A rather than G. Inosine pairs with C rather than T or U. Both alterations can change the amino acid encoded by the mRNA codon where the deamination reaction occurred.

In each case, the nucleotide is modified by removing an exocyclic amine from the six-membered ring and replacing it with a keto oxygen. The change converts C to U and A to I.

The most important properties of living systems are the capacity to catalyze reactions and the capacity to store information that defines the structure of the catalysts. RNA has both of these properties.

5′-AAUAAA and a GU-rich sequence. The 5′-AAUAAA is retained in the mature mRNA.

The 2′, 3′, and 5′ hydroxyls of ribose. The 3′-OH and 5′-OH remain linked to the nucleotides they were bonded to before the reaction. The 2′-OH is linked to the G residue on the 5′ end of the intron.

The function of Dicer is to cleave pre-miRNAs to generate mature miRNAs.

Inactivation of tRNA nucleotidyltransferase. The CCA-3′ would not be added to the tRNA, so amino acids could not attach.

Many naturally occurring ribozymes catalyze reactions involving other RNAs, mainly cleavage or splicing reactions. Ribozymes also catalyze the formation of peptide bonds on ribosomes.

(a) α-Amanitin inhibits Pol II. The rRNA precursors that the researchers wanted to isolate are synthesized by Pol I, and addition of α-amanitin eliminated a lot of background RNA synthesis. (b) Some very stable or tightly bound protein may have remained and catalyzed the intron-splicing reaction. (c) The reaction requires a guanine nucleoside or nucleotide with a 2′-OH (RNA form) and a free 3′-OH. (d) This experiment is a compelling demonstration of self-splicing and RNA catalysis. There are no introns in bacterial rRNA. Expressing the rRNA segment with the use of bacterial enzymes eliminated the possibility that contaminating intron-splicing enzymes from Tetrahymena were present. The Tetrahymena rRNA gene is the only Tetrahymena macromolecule in the reaction mixture and was deproteinized before the experiment.