Biochemical Society Transactions (1999) 27
19
The ThreeDimensional Structure o f Bacterial R i b w m a l F
W
at 13 I! Resolution
Richard Brimacanbe, Wax-Plandc-Institut fur Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany.
Recent advances i n the cryo-electron microscopy of Escherichia
coli ribosomes combined w i t h image processing techniques have led
to computerized reconstructions of these ribosomes a t ever higher
resolution. A reconstruction of 705 ribosomes carrying t R N A a t the
P s i t e and an EF-Tu/tRNA ternary complex stalled w i t h the antibiotic kirromycin at the “pre-A s i t e “ has now been refined to 13 fl
i n the laboratories of Or. W. Wintermeyer (Witten, FRG) and Or. M .
van Heel (Imperial College, London). A t this resolution, the bound
ligands ( t R N A s and EF-Tu) are directly visible i n their entirety,
and furthermore many fine structural elements can be seen i n the
electron density maps which correspond to individual helices of the
rRNA molecules.
Previously, models for the 30 folding of the E. coli rRNA
molecules have been constructed on the basis of the large body of
biochemical data (from cross-linking, foot-printing or similar
studies) that has been accumulated over the years. Now, these models can be combined w i t h the EM density maps to derive the 30
structure of the rRNR i n situ w i t h i n the ribosome. W i t h the help of
a number of new biochemical data sets, including cross-links between the rRNA and mRNA, t R N A and the growing peptide chain, or w i t h i n and between the 55, 165 and 235 rRNA molecules themselves
(collected i n collaboration w i t h the laboratory of Or. 0. Dontsova,
Moscow), we have now fitted a l l three rRNA molecules to the 13 1
EM reconstruction mentioned above. The structure produced satisfies
both the EM density data and the great majority of the biochemical
facts. The X-ray crystallographic structures of t R N A , EF-Tu, and
a number of individual ribosomal proteins ( i n collaboration w i t h
Dr. 5. White, Memphis USA, Or. V . Ramakrishnan, Salt Lake C i t y USA,
Dr. I. Tanaka, Sapporo Japan and Dr. M. Gorlach, Jena FRG) have
furthermore been f i t t e d to the rRNA models i n the 705 ribosome,
again i n such a way as to satisfy both the biochemical and EM dens i t y constraints. The results are giving u s an increasingly accurate
and detailed picture of the structure of the ribosome and of its
interactions w i t h functional ligands.
I10
RNA Crystallography without RNA Crystalls:
Translational Operators, Aptameters and other Motifs
P.Stocklev Department of Biology, Uni of Leeds, Leeds LS29JT
111
A89
Molecular interactions in assembly of
RNA-binding attenuation protein with RN
2
A.A. Antson, E.J. Dodson & G.G. Dodson, York Structural Biology
Laboratory, University of York, York YO1 5DD, UK.
X.-P. Chen & P. Gollnick, Department of Biological Sciences, Cooke
Hall State University of New York at Buffalo, Buffalo, NY 14260,
USA.
The X-ray structure of trp RNA-binding attenuation protein (TRAP)
from B.stearothermophilus in complex with cognate 53-base
(GAGAU),,GAG RNA provides the structural basis for the
transcription regulation of trp (trpEDCFBA) operon.
In several Bacilli, TRAP regulates transcription of the trp operon in
response to changes in the intracellular concentration of L-tryptophan
[ 11. TRAP itself is an assembly of 1 1 subunits related by rotational
symmetry[2]. When TRAP becomes activated by L-tryptophan it
binds specifically to a leader region of the RNA transcript which
contains eleven GAG or UAG triplets[3]. By binding to this RNA,
TRAP disrupts the formation of a hairpin structure (antiterminator)
and allows formation of an alternative, transcription termination
hairpin.
In the structure, each GAG triplet is bound in a pocket formed by pstrands. The circular arrangement of TRAP subunits generates a belt
of 11 GAG binding sites with a diameter of about 80 A. Each RNA
triplet is bound specifically with almost no direct interactions formed
between the sugar-phosphate backbone and the protein.
1 Gollnick P (1994) Regulation of the Bacillus subtilis tr operon by
an RNA-binding protein Molecular Microbiology 11,&I-997.
2 Antson AA, Otrid e J, Brzozowski AM, Dodson El, Dodson GG,
Wilson KS, S m i k TM,Yang M, Kurecki T, Gollnick P The
structure of trp RNA-binding attenuation protein. Nature 1995,
374: 693-700.
3 Babitzke P, Stults JT, Shire SJ, Yanofsky C (1994) TRAP, the rrp
RNA-binding attenuation protein of Bacillus subtilis, is a
multisubunit complex that appears to reco nise G/UAG repeats in
the trpEDCFBA and trpC transcripts. J . dol.Chem. 269: 1659716604.
112 RNA-protein interactions in nuclear pre-mRNA splicing.
L. Verdone,A.E. Mayes, I. Dix and J.D. Beggs
Institute of Cell and Molecular Biology, University of
Edinburgh, King’s Buildings, Mayfield Road, Edinburgh, EH9 3JR,
UK.