Question6

=Text of the Question:= toc Draw the sequence and secondary structure of a ribozyme less than 100 residues in length that cleaves itself to leave a 3' hydroxyl and 5' phosphate. Cite references.

=Possible Answer #1:=

__Sequence and secondary structure:__

"Among group I introns, a quanosine-binding site within P7 activates the 3' hydroxyl for the first trans-esterfication reaction that liberates the 5' exon and links the guanosine substrate to the 5' end of the intron. The exogenous guanosine attacks the phosphorous atom at the 5' splice site of the intron, leaving a free hydroxyl group on the terminal U of the 5' exon (Guo et al. 2004). The second trans-esterfication reaction takes place after a conformational change (facilitated by P10), as the product of the exogenous guanosine exits from the active site and is replaced by the guanosine (ωG) at the 3' terminus of the intron. Then, the hydroxyl group attacks the phosphorus at the 3' splice site, resulting in the ligation of the two exons. The final products are the ligated exons and the excised intron, which has the additional guanosine covalently bound to it's 5' end (Cech 1990; Michel and Westof 1990; Golden et al. 2005). We have found evidence to suggest that this minimal intron goes through this stepwise set of reactions characteristic of group I intron splicing. Sequence evidence from intermediates of the in vitro splicing reactions indicates that the first reaction breaks at the 5' splice site at a GU pair within P1 (Figs. 2,3), often liberating the 5' exon as a 74nt fragment (figs. 1, 2). Furthermore, when the guanosine addid is radioactively labeled almost all of it comigrates within the liberated intron (at 67nt), thus confirming that the first reaction is consistent with group I ribozyme activity."

Harris, Lorena and Rogers Scott O. "Splicing and evolution of an unusually small group I intron". Curr Genet (2008) 54:213?222 DOI 10.1007/s00294-008-0213-y

=Possible Answer #2:=

Among ribozymes, known catalytic RNAs which yield a 5’ phosphate and a 3’ hydroxyl are group I and II introns and ribonuclease P (RNase P).1 The mechanism is also given by Kirsebom:

Figure 6.1: from Kirsebom(2009)1

In this proposed mechanism, RNAs coordinate a metal (most likely Mg+2 or Mn+2) which complexes with a water molecule (shown in violet). The water is activated such that it acts as a nucleophile, attacking the phosphorous of the phosphate group and thus cleaving the 5’ to 3’ phosphodiester bond.

Although a paper was found (Harris 2008) describing a small (67-74nt) group I intron, I did not choose to use this.2 Introns typically are spliced out of a larger transcript and I was almost certain that this exceeded the 100 nt requirement.

In answering this question, I surveyed the RNase P RNAs (RPRs) of several species. The basic idea is to find a minimally catalytically active subset of a ribonuclease P ribozyme (sans protein) and a minimal subset of a targeted substrate it cleaves. These RNAs would then be considered to be a single or composite RNA, thus satisfying the auto-cleaving requirement of the question. RNase P’s which were soley protein or which required complete protein interaction to be catalytically functional were no considered.

While the typical unmatured tRNA (with a 5’ leader needing the be cleaved) would typically be in excess of 80nt, Forster and Altman (1990) investigated smaller subsets of the unmatured tRNA called external guide sequences.3 With these, one could hope to achieve a smaller footprint for the resultant RNA.

The archaea RPRs seemed like a good place to start, since they tended to be shorter and thus would better satisfy the constraints of the question (e.g. < 100 nt total length). Additionally, archaeal RPRs and some baceterial RPRs do exhibit catalytic activity without the additional prosthetic proteins which typically accompany them in living organisms.4 5

Tsai (2006)6 reports that Pyrococcus furiosus RPR (a type A archaea RPR) has 2 domains, an S-domain (for specificity) and a C-domain (for catalytic). The C domain spans nucleotides 1-63, 223-330 and the S domain spans nucleotides 64-222. The secondary structure of the RNA portion of RNase P (e.g. the “apo-ribozyme”) is shown in figure 4a in the paper:

Figure 6.2: RPR of Pyrococcus furiosus (aka Pfu) (from Tsai (2006), fig 4a6)

This has a total length of 330 nucleotides, divided into 171 nucleotides for the C-domain and 159 nucleotides for the S-domain. This is still too long.

Figure 6.3: RPR from E. coli (from JW Brown’s RNase P database)7

According to Green (1996)8, P3, P12, P13, P14, P16, P17 and P18 can be deleted without completely compromising the activity. But the catalytic core of these RPRs are contained in P2,P4, P5 and P15 and all the single strands of RNA linking them.8

Although removing P6, P16-18 destroys in vitro catalytic activity, increasing the amount of ionic solutes available does restore some of the catalytic capability.9 If the catalytic core is shared between E coli and the archaea, then this is consistent with the archaea living in “extreme” environments. This P3 seems to have no direct effect on catalytic ability.9 Indeed, applying these constraints, we achieve a catalytic core of 92nt.

6.4. From7, with 8-9 applied. Catalytic core, 92nt long. UUG is active site, A248 activates.

A mechanism for interaction of the ribozyme with its substrate is given by Kirsebom (1994 and 2009)11, 10:

Figure 6.5: Kirsebom (1994) showing active site and substrate (corresponds with 6.4)

Figure 6.6: From Kirsebom (2009), similar to 6.5, with more detail.

The A248 in figure 6.6 is contained in minimum catalytically active subset shown in 6.4. The tRNA sequence shown in Kirsebom(2009) (also in figure 3a-c) is a full length tRNA which in addition to the minimum catalytic subset would cause us to exceed our length constraint.

However, in Altman and Kirsebom (1999), several subsets of an unmatured tRNA which might interact with this active site are suggested.11 Most significantly a suggested unmatured tRNA subset where there are 11 nucleotides present:

Figure 6.7: (a) from Kirsebom(1999), (b) minimal tRNA for attack.

In figure 6.7, the arrow denotes the cleavage site (when attacked by RNase P). ‘Y’ denote a pyramidine, ‘R’ denotes a purine, ‘N’ means any nucleotide.

From the above discussion, the GG from the active site must be binding with the CCA trailer on the 3’ end of the tRNA. The Y-G on the 5’ of the tRNA subset is where the cleavage actually occurs. The Y on the 3’ end is activated by A248 of the ribozyme (using E coli numbering). This leaves a G with a 5’ phosphate end and the Y leaves with a 3’ hydroxyl end. Since the remaining 4 nt remaining are ‘N’ (don’t care), they must not place a necessary structural role. Hence, I argue that they can be removed, yielding a tRNA subset, which is 7nt long.

The final results is a composite self-catalyzing RNA which is 99nt long and leaves a 5’ phosphate and 3’ hydroxyl, as shown in figure 6.8:

Figure 6.8: Final composite, 99nt. Arrow denotes cleavage site.

1.	[|Kirsebom, L. A. and S. Trobro (2009). "RNase P RNA-mediated cleavage." IUBMB Life. 61(3): 189-200.] 2.	Harris, L.; Rogers, S. O., Splicing and evolution of an unusually small group I intron. Curr. Genet. 2008, 54 (4), 213-222. 3.	[|Forster, A. and S. Altman (1990). "External guide sequences for an RNA enzyme." Science 249(4970): 783-786.] 4.	[|Brown, J. W., E. S. Haas, et al. (1993). "Characterization of ribonuclease P RNAs from thermophilic bacteria." Nucleic Acids Research 21(3): 671-679.] 5.	Pannucci, J. A.; Haas, E. S.; Hall, T. A.; Harris, J. K.; Brown, J. W., RNase P RNAs from some Archaea are catalytically active. Proceedings of the National Academy of Sciences 1999, 96 (14), 7803-7808. 6.	[|Tsai, H. Y., D. K. Pulukkunat, et al. (2006). "Functional reconstitution and characterization of Pyrococcus furiosus RNase P." Proceedings of the National Academy of Sciences of the United States of America 103(44): 16147-16152.] 7.	Brown, J. W., The Ribonuclease P Database. Nucleic Acids Res. 1999, 27 (1), 314. 8.	Green, C. J.; RiveraLeon, R.; Vold, B. S., The catalytic core of RNase P. Nucleic Acids Res 1996, 24 (8), 1497-1503. 9.	Haas, E. S.; Brown, J. W., Evolutionary variation in bacterial RNase P RNAs. Nucleic Acids Res 1998, 26 (18), 4093-4099. 10.	Kirsebom, L. A.; Svard, S. G., Base pairing between Escherichia coli RNase P RNA and its substrate. Embo J. 1994, 13 (20), 4870-6. 11.	Gesteland, R., The RNA World. 2nd ed.; Cold Spring Harbor: 1999.