Metalloribozymes

Although it is apparent that the catalytic parameters of known ribozymes can be substantially altered by mutation and that novel ribozymes can be selected from random sequence populations, there are limits to what the chemistries inherent to the four canonical nucleotides can accomplish. Ribozymes composed only of G, A, U, and C lack acids or bases with pAa values near 7 and have few strong nucleophiles. In consequence, many of the natural RNA catalysts have been shown to be metalloenzymes,...

References

Roberts, J.A. Steitz and A.M. Weiner, Molecular Biology of the Gene. Benjamin Cummings Inc, Menlo Park, CA, 1987. 2. T.R. Cech and B.L. Bass, Annu. Rev. Biochem. 1986, 55, 599. 3. T.R. Cech, Annu. Rev. Biochem 1990, 59. 543. 4. S. Altman, Adv. Enzymol., 1989, 62, 1. 5. F. Noller, V. Hoffarth and L. Zimniak. Science, 1992, 256, 1416. 6. P. Nissen, J. Hansen. N. Ban, P.B. Moore and T.A. Steitz, Science, 2000, 289, 920. 7. P. Walter and G. Blobel. Nature. 1982. 299....

The P4p6 Domain Of The Tetrahymena Group I Selfsplicing Intron

Group I introns, which are defined by a conserved catalytic core and reaction pathway, splice precursor RNAs so that mature ribosomal, transfer, or messenger RNAs can be formed.61 Half of the conserved core in the Tetrahymena thermophila intron is found in an independently folding domain consisting of the base-paired (P) regions P4 through P6 (P4-P6).62 By itself, the P4-P6 domain folds into a structure whose chemical protection pattern is very similar to that seen for the P4-P6 region of the...

Internal Loops

The dominant motif in RNA stem-loops is the A-form helix, the conformation of which was well-understood long before NMR spectroscopy was mature enough to contribute in any way. It is a two-stranded, antiparallel, double helix of indefinite length having geometry so well-known it need not be described here.17 There is no restriction on the nucleotide sequence in either of the two strands of an A-form helix, provided the sequence of the other strand is its Watson-Crick complement. If GU wobble...

In Vitro Selection Of Ribozymes

In vitro selection has been applied to the study of both natural and unnatural ribozymes.8 In vitro selection is governed by the same principles that are operative during natural selection first, a pool of heritable diversity is generated. In the case of natural selection, these are the mutations that arise during replication. In the case of in vitro selection, the pool can be chemically synthesized with the desired degree of randomness. Second, variants within the pool are selected for...

G1093a

Figure 2 Secondary structures of functional sites characterized at high resolution by NMR spectroscopy, (a) The decoding region of 16S rRNA30, indicating sites of mutations, protections by tRNA, and sites of contact with the aminoglycoside antibiotic paromomycin detected by NMR spectroscopy, (b) The dimethyl A stem loop39 and the adjacent 3' terminus of 16S rRNA. The anti-Shine-Dalgarno sequence which base pairs with tnRNA during translation initiation is indicated, (c) The GTPase center of 23S...

J

Figure 14 Sequence of the C domain of PSTV in the rod-like structure (left) and the potential hairpin stem-loop structure that can be formed from it (right). The four arrows represent the inverted repeat sequences.73'74 the base residues of which are numbered. The UV-induced cross-linking between G98 and U260 is shown by a double-headed arrow. (Reproduced by permission of Oxford University Press from Nucleic Acids Res., 1997, 25, 2683.) one clone contained the top strand of the C domain...

Ua

Model substrates for RNase P. (A) pATl, a small model substrate for E. coli RNase P 62 (B) Target RNA-EGS (external guide sequence) complex for E. coli RNase P 21 (C) Small model substrate for human RNase P 9'103 (D) Target RNA-EGS complex for human RNase P.103 a different structure for this loop and many of them lack the GGU sequence. In some instances there is an helix inserted in the loop.20'97 It is interesting that tRNA genes in cyanobacteria do not encode the CCA, suggesting an...

Summary And Perspectives

The discovery that nuclear-encoded mRNAs of metazoa were subject to RNA editing altered the prevailing view that editing was relevant only to those studying mitochondrial gene expression in single-celled eukaryotes. Two types of RNA editing have been found in nuclear-encoded mRNAs, and one type has been observed ubiquitously in every metazoan assayed. The RNA editing observed in nuclear-encoded RNAs involves deamination of encoded nucleotides, rather than the insertion and deletion of...

Data Analysis

Experimental techniques for measuring melting curves have been described in detail.64'66 Given an absorbance vs. temperature melting curve of a duplex to single strand transition like the one in Figure 2, thermodynamic information can be derived by analyzing a as a function of temperature. Under the two-state transition assumption, the measured extinction coefficient, s(T), at any temperature can be expressed as a mole-fraction weighted linear combination of two components, ess and ds 66 where...

P A C

Kcal mol1 less stable at 37 C than GGAG- This enhancement, however, depends on the orientation of the closing base pairs and of the potential GA mismatch in the loop. Loops without potential GA or UU mismatches fall in a relatively narrow range with an average free energy of loop formation of 2.2 kcal mol-1. This trend is similar to the results for symmetric tandem mismatches. The results allow approximations for loops that have not been measured.48'154 These approximations are listed along...

New Approaches

No one familiar with the history of NMR spectroscopy would dare suggest that the NOESY COSY approach just described will turn out to be the only way to determine RNA solution structures, or even the best way. NMR spectroscopy has shown an amazing capacity for growth and renewal over the years, and recent developments in the protein NMR field suggest that improved methods for RNA structure determination will soon be available. NMR has been used to study solids for decades, and in recent years...

University of California Berkeley CA USA

3.2 CRYSTALLIZATION OF 3.2.1 Production of Homogeneous RNA by In Vitro Transcription 3.2.2 Chemical Synthesis 3.2.3 Purification of RNA for Crystallization 3.2.4 Establishing the Suitability of RNA Preparations for Crystallization 3.2.5 Sparse Matrix Approaches to RNA 3.3 HEAVY ATOM DERIVATIVES OF RNA 3.4 DUPLEX STRUCTURES 3.4.1 Metal Ion Interactions in RNA 3.4.2 Noncanonical Base 3.4.3 RNA Packing and Hydration 3.5 TRANSFER 3.6 THE HAMMERHEAD 3.7 THE P4-P6 DOMAIN OF THE TETRAHYMENA GROUP I...

Info

A Abbreviations include k tcat k K kczt Km m.M minutes.molar mt multiple turnover RdO Round 0 rt relative to st single-turnover. b The rates and rate enhancements that are reported are generally taken directly from the primary literature. Usually only one of several rates or an average rate is reported. No attempt is made to reconcile apparently contradictory values (e.g., the differences between estimates for rate acceleration by Conn et al 1 and Li and Sen78 for similar reactions) or to judge...

Identifying Functional Sequences And Structures

A variety of natural ribozymes, ranging from the small hairpin and hammerhead ribozymes to the large Group I self-splicing intron and ribonuclease P, have been segmentally randomized and selected for catalytic function. Data from these selection experiments, such as the locations of functionally important residues and sequence covariations, have been used for the development of ribozyme structural models. We will consider several particularly illuminating examples. The relatively small size...

Watson Crick Helical Regions

Watson-Crick base pairs are the most abundant motif in RNA structures, typically accounting for about 50 of nucleotides in a sequence. They form the helical framework for RNA and are also central to RNA-RNA recognition processes.1'98'99 Therefore, they are the most extensively studied motif. Measurements on the sequence dependence of energetics of Watson-Crick duplex formation by RNA oligonucleotides and polynucleotides show that base pair composition (mole fraction of AU and GC base pairs)...

Coaxial Stacks and Multibranch Loops or Junctions

Coaxial stacking can occur when two helices are directly adjacent or separated by a mismatch. Coaxial stacks have been seen in crystal structures of tRNAs11-13'156'157 and are essential for the three-dimensional shape of tRNA. Stability increments from coaxial stacking of Watson-Crick pairs and of GA or CC mismatches have been investigated in model systems where a short oligomer binds to a four- or five-nucleotide overhang at the base of a hairpin stem.52'158'159 This binding creates an...

Secondary Structure Formation

Formation of double helix requires a nucleation event to bring the complementary regions of the sequence together, so the overall rate of forming a double helix is much slower than the rate of helix growth. If the reaction is bimolecular, the rate is governed by a corresponding rate constant that is typically in the range of 106 to 107 M_1 s-1.23,24 With a strand concentration of 1 iM, the characteristic time for helix formation is thus roughly 100 ms to 1 s. Hairpin helix formation is...

Conclusions

10.9.1 Natural Ribozymes and Selection Structure Determination The benefits of artificial phylogenetic analyses for nucleic acid structure prediction become apparent when RNA and protein catalysts are compared. The primary sequence of both types of biopolymers determines their global architecture, which in turn determines their catalytic functions. However, protein folds are determined partially by the formation of secondary structural elements, and largely by the establishment of tertiary...

Replication Of Viroids

12.6.1 Rolling Circle Mechanism of Replication There seems to be universal agreement that viroids are replicated by a rolling circle mechanism, as first proposed in 198440 (Figure 8), and the finer aspects of their replication are slowly being unraveled. In one of the two variations of the rolling circle mechanism (Figure 8(a)), the infectious plus RNA is copied continuously by an RNA polymerase to produce a long minus RNA strand. Specific cleavage of the ( ) strand, either enzymatically or RNA...

The Rate Of Base Pairing

Formation of a base pair is the elementary reaction step in the transfer of genetic information. Because of the cooperative character of double helix formation, the dynamic and equilibrium properties of base pairs differ greatly depending on whether they are located at the end of a helix or in its interior. Base pairs at the end of a helix open and close rapidly in a process called fraying, which has an equilibrium constant of roughly 2-10, depending on the nature of the base pair and its...

Editing By Base Modification

RNA modifications have long been known to occur in tRNAs, rRNAs, and mRNAs.4'5 Some of these modifications are simple, involving merely the methylation of an existing nucleotide, while others are quite complex, involving multiple enzymes for their synthesis. As additional types of RNA editing are discovered, it becomes harder to distinguish the difference between what has traditionally been called an RNA modification, from what is now categorized as RNA editing. At one point in time the term...

Structure And Evolution Of The Rna Subunit

The RNA subunit of RNase P from Bacteria has been characterized in many strains. The RNase P database,7 maintained by Dr. James Brown (North Carolina State University URL http www.mbio. ncsu.edu RnaseP home.html), contains almost 200 sequences of bacterial RNase P RNAs in addition to representatives of all other main phylogenetic groups. Once the first RNase P RNA genes were identified, additional ones were isolated by heterologous hybridization, analysis of genome databases and PCR...

Other Applications Of Nucleotide Modifications

Alternative strategies to those described above, that also make use of base modifications in RNA, have been successfully applied to investigate both structural and functional aspects of RNA. These include interference experiments, site-selected insertion of defined modified residues and exploiting the presence of enzyme-catalyzed modified nucleotides in natural RNAs. Interference experiments are aimed to identify at once those elements within an RNA that are involved in a functional process...