Historical & Experimental
iii. OVERALL STRUCTURE
iv. SECONDARY STRUCTURE
v. TERTIARY STRUCTURE:
vi. CONFORMATION OF
THE ANTICODON LOOP
IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURE: Molecular Design, Structural Stability and Specificity, Flexibility of the Molecule, Modified Nucleosides & T-Y-C Corner.
INTRODUCTION. Transfer RNA is responsible
for numerous biological function. tRNA is involved in the central role of protein
of enzyme biosynthesis,
of cell wall,
to RNA-dependent polymerase in reverse transcription among other
many functions. The
overall three dimensional conformation of tRNA is a major contributor
to its multi-functionality.
the advent of refined
techniques in isolation and sequencing mRNAs in the 1960s, the deciphering
of the genetic code dictionary was just a matter of time. Within
the first year following the breakthrough (1961),
Marshall Nirenberg and Heinrich Matthaei established that -UUU-
condon specified Phe. In Kim's
Phe_tRNA structure (1978),
the codon triplet that specifies Phenylalanine is -GAA-.
The structure of yeast (Saccharomyces
phenylalanine transfer RNA (sometimes abbreviated Phe_tRNA) is
depicted on the following Chime/RasMolŠ exhibit. The overall
structure of Phe_tRNA interpreted from x-ray diffraction analysis
at a 2.7 Angstroms resolution by Sung-Hou
Kim and colleagues resembles the shape of the letter "L"
to view L shape tRNA>.
The PDB reference code for the yeast is 6TNA,
published in Nov 16, 1978. During the refinement of this model,
bond lengths and angles within each phosphate group and each nucleoside
were constricted, while those for the linkages between
the nucleosides and phosphates were restrained to standard
backbone of Phe_tRNA folds to form one continuous double-helical
arm with the acceptor
stem and the T stem (Red) <Click to view 1st double-helical
A second double-helical arm is formed by D stem
and the anticodon
Each of these extensions is roughly 60 Angstroms in length and
about 20 Angstroms in diameter, while the distance between the
its two ends is 80 Angstroms. Each stem is a right-handed double
helix running antiparallel to each other. The site of peptide
elongation is located at one extreme, the 3'-end (3'-OH of A76 nucleotide) <Click to view 3'-OH of A76 nucleotide>. At the other extreme,
stem is responsible
for codon recognition on the messenger RNA. The T loop,
located at the corner of the "L," is believed to interact
with ribosomal RNA. The overall structure of tRNA minimizes interference
among its functional regions by isolating its functionally important
can be subdivided into four regions: (1) the amino acid stem, where the amino acid residue is carried
by its 3'-OH terminal <Click button to view amino
acid stem (Red)>
(2) the D
frequently contains the modified base dihydrouridine <Click button to view D
(3) the anticodon
contains the anticodon triplet of bases that complementary to
the codon of the mRNA <Click to button view anticodon
(4) T stem containing the pseudoridine
base <Click button to view T
and (5) the variable
is the site of greatest variability in RNA <Click button to view variable
All four helixes have shallow and deep grooves, while the nucleotide residues are tilted away from its helical
the ribose sugar residues are in a 3'-endo
All four stems
in their helical
(Sussman & Kim, 1978) evidenced by the large standard deviation
associated with their helicity (irregularity surpasses the range
of error of 0.1A based on the evidence of a previous Sussman &
Kim publication). Kim, Sussman and colleagues concluded the irregularities
to be real.
but G4-U69 present in the stem are
Watson-Crick types <Click to view G4-U69 base-pair>.
STRUCTURE: Base-base Interaction. Several factors contribute to the stability
of the tertiary structure of tRNA. all contribute to the stability
of tRNA. Base-base
are based on non Watson-Crick tertiary hydrogen bonding between
bases with the exception of
contribute to the stability of the tertiary structure of tRNA.
and stacking between base
pairs, hydrogen bonding between
base and backbone,
spermines and water interactions all contribute to the stability of tRNA.
Base-base interaction are based on non Watson-Crick tertiary hydrogen
bonding between bases with the exception of G19-C56 (Red) <Click
to show G19-C56
(Red) <Click to show G19-C56
non Watson-Crick base-pair>
. Here shown are nine tertiary base-pairs <Click button to view 9
Notice the stacking of the bases from the three dimensional structure
with the exception of five bases (D16,
U47, and A76)
<Click to view non stacking
STRUCTURE: Base-backbone Interaction. The assignment of tertiary hydrogen bonding
between base and backbone involves the assignment of 12 hydrogen bonds based on the criteria
of distance and stereogeometry. Five of the 12 hydrogen bonds involve the O2' hydroxyl groups from the sugar.
STRUCTURE: Backbone-backbone Interaction. Distance and stereogeometry were again
the criteria used to assign hydrogen bonds within the backbone.
All hydrogen bonds under this category involve O2' hydroxyl groups
except for cases where one could assign hydrogen bonds between
O5' and purine
C8 or pyrimidine C6 of the same nucleotide. These CH----O hydrogen bonds are similar
to the dinucleoside phosphates UpA and ApU (Sussman, 1972; Rubin,
1972; Seeman, 1976). The
prevalence of O2' hydroxyl group H-bonds could be the reason behind
RNA's structural nucleic acids functions as found in tRNAs and
ribosomal RNAs. Similarly this also excludes the participation
of DNAs in these processes.
STRUCTURE: Metal-tRNA Interaction. Specific functionally conformation in
tRNAs is achieved via site-specific bound magnesium ions (Fresco,
1966). There are essentially four sites where magnesium hydrates
are bound to tRNA. Two are located at the D loop (Blue) <Click to view D loop>, one in the anticodon loop (Green)
<Click to view Anticodon
and the last one in the between the region formed by residues 8 through 12
(Magneta) <Click to view residue
8-12 magnesium binding site>.
Magnesium metals thus are major contributors to the in stability
of tRNA's loops and sharp turns.
STRUCTURE: Spermine-tRNA Interaction. Polyamines like and also play an important
role in stabilizing tRNA's tertiary conformation (Saikat &
Cohen, 1976). Kim and his colleagues used spermine in the crystallization
Phe_tRNA. They found out two possible sites for spermine binding:
the formed by the and the The second candidate site is formed
by the and the .
spermine and spermidines also play an important role in stabilizing
tRNA's tertiary conformation (Saikat & Cohen, 1976). Kim and
his colleagues used spermine in the crystallization Phe_tRNA.
They found out two possible sites for spermine binding: the deep groove formed by the T stem (Yellow) <Click to view T stem> and the acceptor arm <View Acceptor stem>. The D stem <Click to view D loop> and the anticodon arm (Green)
<Click to view Anticodon
the second candidate site.
STRUCTURE: Water-tRNA Interaction. There are about 60 molecules bound to tRNA. Because of weak electron
densities from these water molecules, the number of molecules
involved in stabilization must be taken with reservation.
the three-dimensional structure of tRNA is stabilized by the sum
of all the above interactions. Base-stacking and base-pairing between the nucleotides primarily stabilize the
arms of the "L". At the junction of the arms of "L,"
base-stacking combined with base-base, base-backbone, and backbone-backbone
help stabilize the sharp turn. Moreover, three of the four stabilizing magnesium
ions are located
at this central region to further stabilize the loops and turns.
The remaining magnesium ion is found at the anticodon stem, presumably stabilizing this functionally
essential region <Click
to view the stabilization due to base stacking>.
OF THE ANTICONDON LOOP. Unlike
the rest of the molecule, hydrogen
not stabilize tRNA's anticodon
tRNA crystal is packed in orthorhombic crystalline form, occupying
20-25% of the lattice's volume. There are basically four small
regions of contact in tRNA. First, an extensive intermolecular
contact occurs at the D
loop. Residues 17, 19 and 20 of one molecule <Click button to display
residues 17, 19 and 20>
are in contact with residues residues
14, 15 and 16
of a second molecule <Click to show residue
14, 15 and 16>,
56 of a third
molecule <Click to show residue
14, 15 and 16>.
These molecule contacts are primarily van der Waals forces. Two
intermolecular hydrogen bonds between O2' of one residue and the
phosphate oxygen of a second molecule's residue 20, and between
O2' of C13 and O4 of D17. Two hydrated Mg2+ further stabilizes
Second, T stem of
one molecule contacts with the acceptor
a second molecule forming a 14° angle. This contact involves
(1) one hydrogen
bond between the O2' hydroxyl group of ribose 5 of one and the
phosphate oxygen of the other's residue 53; (2) and one van der Waals interaction.
The third region
of contact occurs at the anticodon
base of one
molecule stack with same residue of a second molecule .
The fourth molecular
contact occurs at the 3'
end of tRNA.
Residue 76 of the first contacts
residue 37's modified base of the
75 and 76 of the first make contact with residues 28, 29
and 43 of a third molecule.
intermolecular hydrogen bonds and seven total van der Waals' contacts
can be made in this region.
displays four regions of two consecutive bases intercalated with
a third base. Two of these intercalating regions occur where the
T and D stems
contact. The base stacking sequence G19-G57-G18-A58 is generated by the intercalation of
two bases from residues
58 and 57 with two other bases from residues 18 and 19.
The third involves residues 45 and 46 of the variable loop intercalated with the Adenine 9 (G18-A9-G19). The fourth example of
intercalating bases involves residues
8 and 9 and the base triplet C13-G22-G46. The
3' end ribose assumes a C2'-endo puckering (i.e.
IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Molecular design.
The 3' end,
site of amino acid attachment or peptide growth, is separated
from the anticodon
80A. These two functional
region are about 60A away from the T-Y-C conserved sequence, which
is involved in recognition between tRNA and ribosomal RNA. Since all three functionally
important regions may interact with ribosomal RNA and other RNAs
simultaneously it is important that they are physically maximally
separate from each other.
IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Structural stability
a very stable molecule that can easily renature. The high degree stability
is essential for the multi-functionality of tRNA. are stacked
indicating is primarily a stabilizing factor. Further more, specifies
the overall structural framework of tRNA. These base-pairs are
either semi-conserved or conserved in all tRNAs suggesting that
all tRNAs have similar frameworks.
Because of tRNA's
high stability it easily renatures. The high degree stability
is essential for the multi-functionality of tRNA. 71 out of 76
stacked indicating base-stacking
a stabilizing factor. Further more, nine tertiary base-pairs specifies the overall
structural framework of tRNA. These base-pairs are either semi-conserved
or conserved in all tRNAs suggesting that all tRNAs have similar
frameworks. Another stabilizing factor is the ability of the 2' hydroxyl group to extensively
IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Flexibility
of the molecule. Refinement
parameters of rigid groups
such as base, ribose
and phosphate from x-ray diffraction
data illustrates the overall thermal
of these groups. Results indicate unusually high thermal vibration of the acceptor stem and the anticodon arm and suggest that they are more flexible than the rest of the molecule or that
the arms partially unwind. Thus slight movement of the acceptor stem and the anticodon arm during transpeptidation
and translocation within the ribosome is feasible.
IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: Modified nucleosides.
are not essential to conserve the tertiary structure of tRNA,
therefore strongly indicating that these modified bases are responsible
for site recognition of various protein that interacts with tRNA.
A modified 3' end of the anticodon allows only the codon-anticodon triplet base-pairing
IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURES: T-Y-C
sequence G-T-Y-C-G in the T loops
<Click button to view T
is found in all tRNAs participating in ribosome-meditated peptide
elongation. Experimental evidence (Erdmann, 1976) indicates that
the sequence C-G-A-A-C recognize the T loop sequence at the A-site of the ribosome.
However the crystal structure shows that the T loop
and the D
loop is internally
hydrogen bonded and cannot be available for rRNA recognition unless
conformation changes occur. The same crystal structure shows that
tRNA may undergo conformational changes when induced by 5S rRNA
binding. A twist and a bend of 30° on base-pair G19-C56 suggest
that these conformational changes are feasible.