Self-splicing group I introns share several properties with enzymes besides accelerating the reaction rate, including kinetic behavior and specificity. Binding of the guanosine cofactor to the Tetrahymena group I rRNA intron is saturable ( K m < 30 μM) and can be competitively inhibited by 3′-deoxyguanosine. The intron is very precise in its excision reaction, largely due to a segment called the internal guide sequence that can base-pair with exon sequences near the 5′ splice site. This pairing promotes the alignment of specific bonds to be cleaved and rejoined.

Because the intron itself is chemically altered during the splicing reaction—its ends are cleaved—it may seem to lack one key enzymatic property: the ability to catalyze multiple reactions. Closer inspection has shown that after excision, the 414 nucleotide intron from Tetrahymena rRNA can, in vitro , act as a true enzyme (but in vivo it is quickly degraded). A series of intramolecular cyclization and cleavage reactions in the excised intron leads to the loss of 19 nucleotides from its 5′ end.

The remaining 395 nucleotide, linear RNA—referred to as L-19 IVS (intervening sequence)—promotes nucleotidyl transfer reactions in which some oligonucleotides are lengthened at the expense of others. The best substrates are oligonucleotides, such as a synthetic (C) 5 oligomer, that can base-pair with the same guanylate-rich internal guide sequence that held the 5′ exon in place for self-splicing.

The enzymatic activity of the L-19 IVS ribozyme results from a cycle of transesterification reactions mechanistically similar to self-splicing. Each ribozyme molecule can process about 100 substrate molecules per hour and is not altered in the reaction—that is, the intron acts as a catalyst. It follows Michaelis-Menten kinetics, is specific for RNA oligonucleotide substrates, and can be competitively inhibited. The k cat / K m (specificity constant) is 10 3 M -1 S -1 , lower than that of many enzymes, but the ribozyme accelerates hydrolysis by a factor of 1010 relative to the uncatalyzed reaction. It makes use of substrate orientation, covalent catalysis, and metal-ion catalysis—strategies used by protein enzymes.

group I

group I I

group I II

group IV

压缩机和冷凝器

冷凝器和干燥瓶

干燥瓶和膨胀阀

蒸发器和压缩机

There are four classes of introns. The first two, the group I and group II introns, differ in the details of their splicing mechanisms but share one surprising characteristic: they are self-splicing—no protein enzymes are involved.

Group I introns are found in some nuclear, mitochondrial, and chloroplast genes that code for rRNAs, mRNAs, and tRNAs. Group II introns are generally found in the primary transcripts of mitochondrial or chloroplast mRNAs in fungi, algae, and plants. Group I and group II introns are also found among the rare examples of introns in bacteria.

Neither class requires a high-energy cofactor (such as ATP) for splicing. The splicing mechanisms in both groups involve two transesterification reaction steps, in which a ribose 2′- or 3′-hydroxyl group makes a nucleophilic attack on a phosphorus, and a new phosphodiester bond is formed at the expense of the old, maintaining the balance of energy.

These reactions are very similar to the DNA breaking and rejoining reactions promoted by topoisomerases and site-specific recombinases.

20%

30%

40%

50%

算术均数

几何均数

中位数

标准差

方差

均数

众数

中位数

几何均数与中位数

几何均数