INVESTIGATORS

J. Stevens, O. Blixt, J.C. Paulson, and I.A. Wilson(The Scripps Research Institute); T.M. Tumpey (Centersfor Disease Control and Prevention); and J.K. Tauben-berger (Armed Forces Institute of Pathology).

PUBLICATION

J. Stevens, O. Blixt, T.M. Tumpey, J.K. Taubenberger,J.C. Paulson, and I.A. Wilson, “Structure and recep-tor specificity of the hemagglutinin from an H5N1 in-fluenza virus,” Science 312, 404 (2006).

Ribosomes are RNA-based proteinfactories found in all living cells, re-sponsible for translating the geneticinformation encoded in messengerRNA (mRNA) into proteins. The firstx-ray structures of the complete 70Sribosome were determined in 1999 at7.8 Å and in 2001 at 5.5 Å, using dif-fraction data collected at ALS Beam-line 5.0.2. These structures showedhow the ribosomal RNA and the morethan 50 ribosomal proteins are organ-ized to form the structure of the com-plete ribosome and the positions ofthe mRNA and transfer RNAs (tRNAs)in the ribosome. Now, using data col-lected at ALS Beamline 12.3.1, re-searchers from UC Santa Cruz havesolved the structure of a Thermus ther-

mophilus 70S ribosome functionalcomplex at 3.7-Å resolution (Figure 3).Because of the large cell dimensions

of ribosome crystals, they diffractweakly, and spots are crowded closetogether in the diffraction patterns.

Science Highlights : : Structural Biology

STRUCTURE OF THE COMPLETE 70S RIBOSOME AT 3.7-Å RESOLUTIONThe ribosome is the largest asymmetric macromolecular complex for which an atomic structure has been determined. Thefirst all-atom structure of a ribosome was obtained by Cate and co-workers in 2005, using ALS data, for two differentconformations of vacant Escherichia coli 70S ribosomes at 3.5-Å resolution. Comparison of the 3.7-Å structure of thetRNA-containing 70S ribosome complex with that of the vacant ribosome provides insight into the structural changes thatoccur upon binding tRNA. This has an important bearing on our understanding of the structural dynamics of the ribosomeand tRNA during protein synthesis. Besides providing a sound structural foundation for attempting to understand the molec-ular mechanisms of protein synthesis, the new structure of a functional ribosome complex, together with other structures of70S ribosomes and ribosomal subunits, can be used to understand the molecular basis of action of the many antibioticsthat target bacterial ribosomes, leading to rational design of novel antibiotics.

a b c

FIGURE 3. X-ray crystal structure of a 70S ribosome functional complex at 3.7-Å resolution. Thecomplex contains a defined mRNA (green) and two tRNAs, bound to the P and E sites (orangeand red) at the subunit interface. (a) The 30S subunit is shown on the left (16S rRNA in cyan,30S proteins in dark blue) and the 50S subunit is on the right (23S rRNA in gray, 5S rRNA ingray-blue, and 50S proteins in magenta). (b) The 30S subunit. (c) The 50S subunit.

FUNDING

National Institutes of Health and Skaggs Institute forChemical Biology.

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Science Highlights : : Structural Biology

Consequently, the high-flux beams,sensitive large-area detectors, andwell-focused, compact beam crosssections available at the ALS allplayed a crucial role in this work. Re-search in this area may lead to novelantibiotics targeting bacterial ribo-somes that have developed resistanceto current drugs.

The improved structure resolutionallows construction of the first all-atom model of a ribosome functionalcomplex containing its mRNA andtRNA substrates. This provided twokinds of information crucial to the un-derstanding of the protein synthesismechanism: (1) details of molecularinteractions between the ribosomeand its substrate RNAs, and (2) waysin which the structures of both the ri-bosome and the tRNAs are altered bytheir functional interactions.

During protein synthesis, the ge-

netic code is used to translate the se-quence of nucleotides in an mRNAinto the sequence of amino acids inthe protein. Groups of three nu-cleotides (codons) in the mRNA areread by base pairing with a comple-mentary three-nucleotide sequence ofnucleotides (anticodon) in the tRNAs,which carry the growing proteinchain during synthesis. There arethree binding sites for tRNA in the ri-bosome, called the A (aminoacyl), P(peptidyl) and E (exit) sites. In thecrystals that were used for the struc-ture determination, tRNAPhe wasbound to the P site and noncognateendogenous tRNA to the E site of theribosome in addition to a 10-nu-cleotide mRNA fragment. The tRNAis bound most tightly to the ribosomalP site, which is responsible for main-taining the translational readingframe of the mRNA and for holding

the growing protein chain in the ribo-some via the peptidyl-tRNA. Thestructure reveals a high density ofcontacts between the tRNA and ribo-somal RNA bases and backbone, aswell as ribosomal proteins, explainingthe high affinity of the P site fortRNA (Figure 4). Catalysis of peptidebond formation takes place on the 50Ssubunit by a ribosomal activity calledpeptidyl transferase. In the new struc-ture, an intriguing structural re-arrangement is observed in thepeptidyl transferase center (Figure 5).

In the course of protein synthesis,tRNAs move through the ribosome,coupled to movement of mRNA likean assembly line, in a process calledtranslocation. Translocation of tRNAfrom the P to the E site is crucial forthe energetics of this process, and re-quires that the terminal nucleotideA76 of tRNA is deacylated—i.e., no

G1338

m22G966

m5C1400

m3U1498

m4Cm1402

A1339

C1403

A790

S9

S13

FIGURE 4. Interactions of the anticodon stem loop of elongator tRNAPhe (orange) and mRNA(green) with 16S rRNA (cyan) and small-subunit proteins (blue) in the 30S subunit P site.

C2573

A2602

A2451A2450

C2064

FIGURE 5. Structural changes in the peptidyltransferase center. View of the T. ther-mophilus 70S ribosomal complex containingdeacylated tRNA bound to the P site (blue)compared with the H. marismortui 50S sub-unit model complex (magenta).

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longer contains a bound proteinchain, which is its chemical state fol-lowing peptide bond formation. Thenew structure explains this require-ment, showing that binding of tRNAto the E site requires hydrogen bond-ing between the ribose moiety of A76and C2394 of 23S rRNA (Figure 6);the presence of a peptidyl, aminoacyl,

or even a methyl group bound to theterminal ribose would thus preventthese interactions.

Future ribosome structure studiesthat include functional states mayeventually lead to an atomic-resolu-tion three-dimensional “movie”—theultimate description of the molecularmechanism of protein synthesis.

INVESTIGATORS

A. Korostelev, S. Trakhanov, M. Laurberg, and H.F.Noller (UC Santa Cruz).

PUBLICATION

A. Korostelev, S. Trakhanov, M. Laurberg, and H.F.Noller, “Crystal structure of a 70S ribosome-tRNAcomplex reveals functional interactions and rearrange-ments,” Cell 126, 1065 (2006).

FUNDING

National Institutes of Health and Agouron Institute.

Science Highlights : : Structural Biology

a b

L1 stalk C2394

G2421

A2422

L28

E-tRNA

C74C75

C76

FIGURE 6. E-site tRNA interactions. (a) Interaction of the elbow of E-site tRNA (red) with 23S rRNA (blue) in the L1 stalk region, showing the large-scale displacement of the stalk relative to its position in the vacant ribosome, induced by tRNA binding. The blue arrow indicates the extremecompression of the major groove of helix 76 of 23S rRNA that accompanies this movement. (b) Interactions of the CCA tail of E-site tRNA withthe large subunit.

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