Damage Selection Experiments with Ribozymes

A wealth of information has been accumulated on ribozymes since their discovery nearly 20 years ago. Most of the studies can be fitted into one of the three main lines of research: (1) characterization of known ribozymes (that is, inferring the structure and mechanisms of catalysis; Lilley, 1999); (2) modification of natural ribozymes to be used in therapeutics (Sullenger and Gilboa, 2002); and (3) in vitro evolution of novel ribozymes (Joyce, 1998, 2002; Landweber et al., 1998; Spir-in, 2002). The characterization of ribozymes frequently involved mutagenesis experiments, where the enzymatic activity of certain mutants was measured in order to get insight into either the structure of the molecule or the mechanism of catalysis. While not directed toward the study of fitness landscapes, these experiments certainly contain a wealth of empirical information necessary for assembling the realistic fitness landscape of the studied ribozyme. Albeit all naturally occurring ribozymes are being studied extensively, there are only a few instances where the realistic fitness landscape can be conveniently investigated. Group I and group II introns, as well as the RNAase P, have to be excluded because of their rather large size. Furthermore, it is inevitable to employ an RNA folding algorithm in any sensible investigation of the fitness landscapes of ribo-zymes. Therefore, ribozymes with a pseudo-knot in their structure also have to be excluded because most conventional folding algorithms cannot satisfactorily cope with pseudo-knots. This requirement singles out the hepatitis delta virus, which contains such structural elements (Perrotta and Been, 1991).

On the other hand, the hammerhead, hairpin, and Neurospora VS self-cleaving ribozymes can be separated into a substrate and a trans-cleaving ribozyme. With respect to these three ribozymes the trans-cleaving enzyme does not contain a pseudo-knot structure. The hammerhead can be separated into a 13-mer enzyme and a 41-mer oligonucleotide substrate (Jeffries and Symons, 1989). The hairpin can be separated into a 50-mer enzyme and a 15-mer substrate (Fedor, 2000). The trans-acting ribozyme is 144 nucleotides long for the Neurospora VS ribozyme (Fig. 3.2), and the substrate is 20 nucleotides long (Guo and Collins, 1995). We restrict our further analysis to the trans-acting ribozyme, and assume that the substrate is the same as the natural one. Unfortunately for our study, many of the mutagenesis experiments have been directed towards the substrate and substrate-binding regions (Joseph et al., 1993; Joseph and Burke, 1993; Nishikawa et al., 1997; Ananvoranich and Perreault, 1998; Perez-Ruiz et al., 1999) in order to produce new RNA- or DNA-cleaving ribozymes to be used in therapeutics (Yu et al., 1998; Andang et al., 1999; Andang et al., 2004; Zhang et al., 2004). Based mainly on experiments with the VS and the hairpin ribozymes the following general conclusions can be derived:

• Structure is important. From experimental data on the VS ribozyme Lilley and co-workers (Lafontaine et al., 2002c) state that "the secondary structure of the ribozyme is important, but the nature of most individual base pairs is not. Many can be reversed or replaced by a different pair without major loss of activity, so long as a base pair is retained at a given position." Similarly, in the hairpin ribozyme all base pairs can be altered (except base pair G11:C/U-2) as long as the base pairing is maintained (Fedor, 2000).

• There are critical regions in the molecule. For the single-stranded regions the structure has to be maintained, but at many such positions the nature of the base located there is also important. For example, most of the bases in the four loops of the hairpin ribozyme are essential, and any change in those positions severely reduces activity (Siwkowski et al., 1997; Shippy et al., 1998). For the VS ribozyme 16 such critical sites were identified (Lafontaine et al., 2002a): these sites are located around the active site, the substrate-binding region, and in the two- or three-way junctions.

• Structure can be varied slightly. The structure of the naturally occurring ribozymes can be slightly varied, as there are regions that are not crucial to function. For example, the stem-loop IV of the VS ribozyme is virtually completely dispensable, but the junction 3-4-5 must be formed (albeit after the complete removal of stem-loops IV and V the ribozyme still has detectable activity; Sood and Collins, 2002). Similarly, in the hairpin ribozyme the helices H1 and H4 can be shortened and greatly extended without any loss of activity (Fedor, 2000; Sargueil et al., 1995).

Fig. 3.2 Sequence and secondary structure of the enzyme part of the Neurospora VS ribozyme (numbering according to Beattie et al., 1995). Roman numbers indicate the regions of the ribozyme. Capitalized nucleotides indicate positions for which mutagenesis studies are available. Bold nucleotides indicate critical sites.

Fig. 3.2 Sequence and secondary structure of the enzyme part of the Neurospora VS ribozyme (numbering according to Beattie et al., 1995). Roman numbers indicate the regions of the ribozyme. Capitalized nucleotides indicate positions for which mutagenesis studies are available. Bold nucleotides indicate critical sites.

While the previous general conclusions can be easily incorporated into a model of a fitness landscape, one general difficulty stills remain; namely, the combined effect of multiple mutations. Most mutagenesis experiments have investigated only single mutations (or mutations involving a base pair) in the vicinity of the wild type in sequence space and rarely report the activity of double or higher order mutants. In those few instances where the effects of multiple mutations were evaluated, the activities of the single-point mutants were not always included. The only remarkable exception is the study of Lehman and Joyce (1993) from an initial pool of the Tetrahymena ribozyme, where they found that in general the mutational effects were multiplicative (which implies mutational additivity for ribozyme activity).

Table 3.1 summarizes the available experimental information on multiple mutational effects for some of the known nucleolytic ribozymes. The plot of the measured enzymatic activities of the double mutants on the estimated activities from the single mutants (Fig. 3.3) clearly suggests that mutational effects are nearly multiplicative, with a slight positive synergy. Such positive synergy was also found for chemical modifications of the hairpin ribozyme (Klosermeier and Millar, 2002). Accordingly, the fitness of a molecule containing n mutations (wn) could be estimated as:

multiplicative , b\

where wi is the fitness of a single-error variant, wnmultiplicative is the fitness of an n-error variant assuming multiplicative effects, and a and 0 are parameters to be fitted given the data (Fig. 3.3). We stress, however, that although the data set contains information from three different ribozymes the number of points is still quite small. Therefore, some care should be taken when translating the empirical available information to a fitness function.

Besides these synergistic effects there are also examples of mutations that "rescue" enzymatic activity to some extent. Mutations that result in the loss of catalytic activity also exist (Table 3.2). Mutants containing these and other point

Fig. 3.3 Plot of the measured enzymatic activities of the double mutants on the estimated activities from the single mutants for the Neurospora VS ribozyme.
Table 3.1 Available experimental data for the evaluation of multiple mutant effects on fitness

Mutant 1

Activity

Mutant 2

Activity

Activity of the double mutant

Estimated activity of the double mutant

Reference

U39Ca

0.289

A11G

0.32

0.093

0.095

Joseph et al., 1993

G21Ua

0.01

A20U

0.9

0.01

0.009

Sargueil et al., 2000

G21Ua

0.01

A20G

0.2

0.003

0.002

Sargueil et al., 2000

G21Ua

0.01

A20C

0.6

0.15

0.06

Sargueil et al., 2000

G21Ua

0.01

A43G

0.4

< 0.001

0.004

Sargueil et al., 2000

G21Ua

0.075

A43G

0.085

< 0.001

0.0064

Siwkowski et al., 1997;

Sargueil et al., 2000

G21Ua

0.075

A43U

0.002

< 0.001

0.00015

Siwkowski et al., 1997;

Sargueil et al., 2000

A7Ca

1

A20C

0.81

1.04

0.81

Anderson et al., 1994

A730Cb

0.32

A731C

0.39

0

0.12

Kumar et al., 1992

G726Ab

0

A730C

0.32

0

0

Kumar et al., 1992

U752Cc

0.80

U753C

0.42

0.52

0.336

Lafontaine et al., 2001b

G722C;

0.81

C723G;-

0.84

0.75

0.680

Beattie et al., 1995

C763Gc

G762C

G716Cc

0.21

U717A

0.21d

0.02

0.044

Beattie et al., 1995

C662Gc

0.23

A661U

0.23d

0.06

0.053

Beattie et al., 1995

a Hairpin ribozyme (numbering follows Butcher and Burke, 1994a,b). b Hepatitis delta virus (numbering according to Makino et al., 1987). c Neurospora VS ribozyme (numbering according to Beattie et al., 1995). d No data available. The activity of mutant 2 is assumed to be equal to the activity of mutant 1.

a Hairpin ribozyme (numbering follows Butcher and Burke, 1994a,b). b Hepatitis delta virus (numbering according to Makino et al., 1987). c Neurospora VS ribozyme (numbering according to Beattie et al., 1995). d No data available. The activity of mutant 2 is assumed to be equal to the activity of mutant 1.

mutations might or might not have detectable activity. To our knowledge these interactions are impossible to predict, thus they can only be incorporated into a definition of a fitness landscape if known from experiments. In conclusion, the easiest way to deal with multiple mutations is to assume mutational independence (multiplicative effects), although it slightly overestimates the decrease in fitness due to multiple mutations. A more realistic assumption can come from taking the synergy into account, albeit more data would be highly welcome. If rescue mutations or other such effects are known of the ribozyme, then they can also be incorporated to increase the realism of the fitness landscape.

Table 3.2 Single null and multiple mutations that in some cases "rescue" enzymatic activity to some extent

Mutation that abolishes activity

Multiple mutant

Relative activity

Ribozyme

Reference

A43C

A43C; G21U

0

Hairpin

Siwkowski et al., 1997a; Sargueil et al., 2000

G726A

G726A; A730C

0

HDV

Kumar et al.,

1992

G726A

G726A; G727C; A731C

0.02

HDV

Kumar et al.,

1992

G726A

G726A; G728A; C729G

0

HDV

Kumar et al.,

1992

G726A

G726A; G727U; C729G

0.01

HDV

Kumar et al.,

1992

G726C

G726C; G728U; A730C

0

HDV

Kumar et al.,

1992

G726U

G726U; G727U; A731C

0.04

HDV

Kumar et al.,

1992

G727C

G726A; G727C; A731C

0.02

HDV

Kumar et al.,

1992

G727C

G727C; C729A; A731G

0

HDV

Kumar et al.,

1992

G728C

G727A; G728C

0.03

HDV

Kumar et al.,

1992

G728C

G728C; A731U

0.01

HDV

Kumar et al.,

1992

G728C

G728C; A730G

0.06

HDV

Kumar et al.,

1992

G728C

G727A; G728C; C729U

0.04

HDV

Kumar et al.,

1992

C763A

C763A; A766G

0

HDV

Kumar et al.,

1992

C763A

C763A; G764A

0

HDV

Kumar et al.,

1992

C763G

C763G; A765U

0

HDV

Kumar et al.,

1992

C763G

C763G; A765U

0

HDV

Kumar et al.,

Turbo Charged Fitness With The Tabata System

Turbo Charged Fitness With The Tabata System

The Tabata workout system is a version of the High Intensity Interval Training program developed by Professor Izumi Tabata as training for Olympic speed skaters in 1996. The results studies conducted on the training program confirm that even a four minute cardiovascular exercise routine improves a persons level of fitness.

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