Introduction.
This section describes a few of the more interesting methods currently used to study the secondary structure of RNA molecules.
Some of these methods, especially in combination with molecular modeling, can give insight into tertiary (three-dimensional)
structure.
A. Chemical and Enzymatic Analysis
Enzymes and chemical agents that cleave or modify RNA with specificity can be used to analyze the structure of RNA. Nucleases
used in RNA structure analysis exhibit single- or double-stranded specificity and some nucleases have sequence preference.
Nucleases used commonly for structural analysis are shown in the table.
Table
1. Nucleases used for RNA Secondary Structure Elucidation.
|
Enzyme
|
Single- or double-stranded
|
Sequence requirement
|
|
S1 nuclease
|
single-stranded
|
none
|
|
RNase T1
|
single-stranded
|
guanosine at 5'
|
|
RNase V1
|
double-stranded
|
none
|
Chemical agents shown in the table are used to modify an RNA residue at a specific
position. An RNA chain can be cleaved at a position of chemically modified base by aniline treatment.
Chemical
Reagents Used for RNA Secondary Structure Mapping.
|
Chemical agents
|
Specificity
|
|
DMS (dimethyl sulfate)
|
N-7 guanosines and N-3 cytosines that are not involved in Watson-Crick base pairs.
|
|
DEPC (diethyl pyrocarbonate)
|
N-7 adenosines that are not involved in tertiary (e.g., Hoogsteen) interactions.
|
|
CMCT (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate)
|
N-1 guanosines and N-3 uracils that are not involved in tertiary interactions.
|
Chemical and enzymatic probes often provide limited information about RNA structure.
Because an RNA molecule is folded in a specific manner, some residues may not be accessible to chemical or enzymatic probes.
In some cases, chemical or enzymatic digestion is carried out at different temperatures. At high temperatures, an RNA molecule
may unfold so that the probe can reach the residues that were not accessible previously. Change in digestion pattern at
different temperatures may provide additional information regarding the tertiary interactions.
B. Nucleotide Analog Interference Mapping.
RNA structure and function can be analyzed with nucleotide analogs. A nucleotide analog may have: 1) a functional group
deleted; 2) a new atom attached (i.e.. F-); and 3) an additional bulky group attached (CH3
group). Nucleotide analogs can be incorporated randomly into an RNA of interest and their effect can be studied.
|
|
Figure 10. Structure of a probe for function of adenine N6.
|
Nucleotide analogs are tagged with α-phosphorothioate (a sulfur atom, S-, is attached to
where an oxygen atom occupied previously). Iodine is used to cleave an RNA selectively at the incorporation site by cleaving
the phosphorothioate linkage. It is necessary that the experiment must be adjusted so that only 1 nucleotide analog is incorporated
per 1 RNA molecule. Because phosphate oxygens are often involved in coordination of cations in catalytic RNAs, the effect
of phosphorothioate alone can also be studied. An example of nucleotide analog is shown in the figure. N-methyladenosine
contains a bulky CH3
group. The methyl group may disrupt a tertiary interaction by steric hindrance or preventing hydrogen bond formation.
Application to RNase P RNA
Phosphorothioate modification-interference is used to probe phosphate oxygens that are involved in the catalysis of RNase
P RNA. RNase P RNA is a ribozyme that catalyzes the cleavage of precursor transfer RNA to generate a transfer RNA with a
mature 5'-end. The reaction requires the presence of divalent ions such as magnesium ions (Mg2+). In order to
observe the effect of phosphorothioate substitution, active ribozymes must be separated from inactive ribozymes. Because
RNase P catalyzes the reaction intermolecularly (RNase P works on a separate molecule), RNase P-pre tRNA conjugate was used.
In this system, RNase P catalyzes a self-cleavage reaction to release a mature tRNA from the conjugate molecule. Therefore,
in an electrophoretic gel, the active ribozyme produces two fragments and the inactive ribozyme produces no fragments. Substitution
of the critical phosphate oxygens to sulfurs interferes the reaction, producing no fragments. Substitution of phosphate
oxygens that are not involved in catalysis has no effect, producing two fragments. By this method, it was possible to identify
four phosphate oxygens that were important in catalysis. The interference of one phosphorothioate substitution is rescued
in the presence of manganese ions (Mn2+), producing normal catalysis. Because the sulfur has a preference for
Mn2+ ions instead of Mg2+ ions, the result indicates the phosphate oxygen at this position is likely
to coordinate a magnesium ion directly.
C. Phylogenetic Analysis (Comparative Structure Analysis)
Phylogenetic analysis is a very powerful method to determine the secondary or in some cases, a tertiary structure of RNA.
First, sets of homologous sequences from diverse organisms are aligned using conserved sequences as markers. Next, a number
of covariation within the homologous sequences are searched. The term, covariation, refers to residues that vary in concert.
For example, if two residues change in concert for a number of organisms and the changes always preserve the Watson-Crick
base pairing, this implies that a physical contact is likely to occur at this site. If there is a stretch of complementary
sequences in the homologous sequences and there are a number of covariations ( about 4 covariations) found in these sequences,
then there is a strong possibility that these sequences may form a helix (stem) structure. By this method, the presence
of helices and RNA-RNA interaction can be predicted. In phylogenetic analysis, we are taking advantage of nature's own mutational
experiment. If a stem structure is important for the overall stability or its function, then, those mutations that have
survived in various organisms must confer the rules of the Watson-Crick or canonical interactions.
The secondary structure proposed by this method can be compared with results from
chemical and enzymatic study or site-directed mutagenesis can be performed to confirm the specific structures proposed by
the phylogenetic analysis. Phylogenetic analysis has been used to determine the secondary structures of various RNAs, including
tmRNA and recently, telomerase RNA.
Methods to study RNA structure and function
1. Brown, J. W., Nolan, J. M., Haas, E. S., Rubio, M. A. T., Major, F., and Pace, N. R. Comparative analysis of ribonuclease
P RNA using gene sequences from natural microbial populations reveals tertiary structural elements. Proc. Natl. Acad.
Sci. USA (1996) 93, 3001-3006.
2. Felden, B., Himeno, H., Muto, A., McCutcheon, J. P., Atkins, J. F., and Gesteland, R. F. Probing the structure
of the Escherichia coli 10Sa RNA (tmRNA). RNA (1997) 3, 89-103.
3. Harris, M. E., Nolan, J. M., Malhotra, A., Brown, J. W., Harvey, S. C., and Pace, N. R. Use of photoaffinity
crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA. EMBO J. (1994) 13,
3953 -3963.
4. Harris, M. E. and Pace, N. R. Identification of phosphate involved in catalysis by the ribozyme RNase P
RNA. RNA (1995) 1, 210-218.
5. Muto, A., Ushida, C., and Himeno, H. A bacterial RNA that functions as both a tRNA and an mRNA. Trends
Biochem Sci (1998) 23, 25-29.
6. Pace, N. R., Thomas, B. C., and Woese, C. R. Probing RNA structure, function, and history by comparative
analysis. The RNA World, Second Ed. Cold Spring Harbor Laboratory Press (1999)
7. Strobel, S. A. A chemogenetic approach to RNA function / structure analysis. Curr Opin Struct Biol
(1999) 9, 346-352.
8. Williams, K. P. and Bartel, D. P. Phylogenetic analysis of tmRNA secondary structure. RNA (1996)
vol. 2, 1306-1310.
9. Zuker, M. Computer Prediction of RNA Structure Methods Enzymol. (1989) 180, 262-288
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