DNA
Function:
Information storage - very stable
Structure:
Usually duplex, B-form
RNA
Function:
Information storage and transport (mRNA, viral
RNA)
Info transfer (tRNA)
Catalysis -ribozymes
Ribosome
Regulation - Plasmid copy #, attenuation
Structure:
"Single-stranded" But actually lots of intramolecular
base pairing to form globular arrangement of short helical structures.
Primary : - Nucleotide sequence from RNA or DNA
Secondary: - Watson Crick base pairs
- Comparative analysis
- Chemical and Enzymatic probes
- Computer predictions
Tertiary: - H-bonds, stacking
- Crystallography
- NMR (very few examples)
- Comparative analysis
- Crosslinking
Phylogenetic Comparative Analysis:
Sequence homologous RNAs from a variety of organisms
Align sequences
Look for covariation of base sequences as evidence of interaction
(i.e. W-C or other pairs)
|-|
U<-- A-U -->A
|-|
A<-- C-G -->U
|-|
Complementarity maintained by compensatory base
changes.
Can also detect some non Watson-Crick tertiary interactions this way.
(Computer programs aid in aligning seqs. and detecting covariations)
Also identifies highly conserved sequences that may be required for
special functions.
BUT: Cannot tell base-pairing of conserved residues that don't
vary
Examples of well-defined secondary structures
derived from phylogenetic analysis:
tRNA
rRNA (16S, 23S, 5S)
Group I intron
Group II intron
RNase P
| |
|
Chemical and Enzymatic probes:
• (see Ehresman (1987) NAR15, 9109 for review)
Provide info on secondary structure and accessibility
of nucleotides
(e.g. RNase T1, T2 attack only unpaired bases, V1 only attacks paired)
(kethoxal, DMS attack base paired face of G, A only when not paired)
Results can be difficult to interpret, though.
|
Probe |
MW |
Specificity |
|
RNase T1 |
11,000 |
unpaired G |
|
RNase U2 |
12,500 |
unpaired A>G |
|
RNase CL3 |
16,800 |
unpaired C>A>U |
|
RNase T2 |
36,000 |
unpaired N |
|
S1 nuclease |
32,000 |
unpaired N |
|
N. c. nuclease |
55,000 |
unpaired N |
|
RNase V1 |
15,900 |
paired or stacked N |
|
DMS |
126 |
N3-C |
|
DEPC |
174 |
N7-A |
|
CMCT |
424 |
N3-U |
|
kethoxal |
148 |
N1, N2-G |
|
bisulfite |
104 |
Unpaired C->U |
|
ENU |
117 |
Phosphate |
|
Fe(II)-EDTA |
Backbone |
Computer prediction based on sequence
Not as straightforward as you would think
Look for regions of molecule that can form W-C pairs with a given region.
But one region of RNA may be complementary to many other short stretches
of sequence.
Computer algorithms can help see which folding
choices yield lowest free energy. (Zuker, 1989)
e.g. G-C pair more favorable than A-U
G•U (wobble) pair is almost as favorable as A-U
(makes for even more regions of complementarity)
Also takes into account "nearest neighbor" rules for stacking:
So identical nucleotide compositions can have different energies due
to order of sequence.
Unpaired structures are unfavorable (loops,
bulges).
But some are more unfavorable than others.
Even with all of this info, still not always very accurate:
Many regions of molecule not involved in W-C pairs, form "non-canonical"
pairs instead: e.g. G-A, A-C, etc.
Non-canonical pairs can have very favorable energy, but many don't tend
to fit A-form helix very well.
Phylogenetics can sometimes predict non-canonicals
Many predicted non-canonical pairs have been confirmed by crystal structures.
Also 2'OH is often involved in tertiary pairs, but it is difficult to
predict, since it is both an H-bond donor and acceptor.
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Self-complementarity |
Computer prediction |
Phylogenetic |
Tertiary Structure:
Crystallography: yields very good structure models, RNA crystal structures becoming more common, even up to ribosome!
(Quigley and Rich, 1976)
Shows very condensed 3° structure:
Arms of cloverleaf stack into helices
loops interact to form L-shaped global structure
U-turn motif: tight turn in anticodon and T-loops
(found again in many other structures)
(Pley et al., 1994)(Scott, 1995)
Also compact structure - 2 of 3 helices stacked
other separated by U-turn to form active site
(Cate et al., 1996; Golden et al., 1998)
New Structural motifs:
Adenosine platforms
Tight turn
Tetraloop acceptor
Ribose Zipper
HDV ribozyme
(Ferre et al., Nature 395 pp. 567 (1998))
Ribosome
High resolution structures of subunits available,
lower resolution structure of intact ribosome. Proteins mostly at periphery
No novel structural motifs, but active site shown to be RNA only
Link
to 30S PDB file
30S subunit: (Nature
2000 Sep 21;407(6802):327-39) a) and b):color-coded RNA. c)
and d) RNA in gray, protein in purple
Large subunit
(Science.
2000 Aug 11;289(5481):905-20.)
NMR
used to determine structure of short sequences (usually <20 nts.)
Computer modeling:
RNase P secondary structure and crosslinks(Chen
et al., 1998)
LeClerc, 1994
Take info from comparative analysis plus other constraints:
Crosslinks
Fluorescence energy transfer
Use as constraints for modeling
Modification interference experiments
(Strobel and Shetty, 1997; Szewczak et al.,
1998)
Identify sites of random incorporations of nucleotide analogs that interfere
with activity of an RNA
Literature:
*=Required reading
*Dickerson et al. (1982) Anatomy of A-, B-, and Z-DNA. Science 216, 475-485.
*Quigley, G. J. and A. Rich. (1976). Structural
domains of transfer RNA molecules. Science. 194, 796-806.
Baskerville, S. and A. D. Ellington. (1995). RNA structure: describing
the elephant. Curr. Biol. 5, 120-123.
Cate, J. H., A. R. Gooding, E. Podell, K. Zhou, B. L. Golden, C. E. Kundrot,
T. R. Cech, and J. A. Doudna. (1996). Crystal structure of a group I ribozyme
domain: principles of RNA packing. Science. 273, 1678-1685.
Chen, J. L., J. M. Nolan, M. E. Harris, and N. R. Pace. (1998). Comparative
photocross-linking analysis of the tertiary structures of Escherichia
coli and Bacillus subtilis RNase P RNAs. EMBO J. 17,
1515-1525.
Ferre, D. A., K. Zhou, and J. A. Doudna. (1998). Crystal structure of
a hepatitis delta virus ribozyme [see comments]. Nature. 395, 567-74.
Golden, B. L., A. R. Gooding, E. R. Podell, and T. R. Cech. (1998). A
preorganized active site in the crystal structure of the Tetrahymena ribozyme
. Science. 282, 259-64.
Pley, H. W., K. M. Flaherty, and D. B. McKay. (1994). Three-dimensional
structure of a hammerhead ribozyme. Nature. 372, 68-74.
Strobel, S. A. and K. Shetty. (1997). Defining the chemical groups essential
for Tetrahymena group I intron function by nucleotide analog interference
mapping. Proc Natl Acad Sci U S A. 94, 2903-8.
Szewczak, A. A., D. L. Ortoleva, S. P. Ryder, E. Moncoeur, and S. A. Strobel.
(1998). A minor groove RNA triple helix within the catalytic core of a
group I intron. Nat Struct Biol. 5, 1037-42.
Zuker, M. (1989). On finding all suboptimal foldings of an RNA molecule.
Science. 244, 48-52.
LeClerc et al (1994) A 3-D model of the Rev-binding
element of HIV-1 derived from analyses of aptamers. Nature Structural
Biology 1, 293-300.
Scott, W. G., J. T. Finch, and A. Klug. (1995). The crystal structure of
an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage.
Cell. 81, 991-1002.
