Molecular Cell

Article

Ribosomal Protein L3: Gatekeeper to the A SiteArturas Meskauskas1,* and Jonathan D. Dinman1,*1Department of Cell Biology and Molecular Genetics, University of Maryland, Microbiology Building, Room 2135, College Park,

MD 20742, USA

*Correspondence: artmeska@wam.umd.edu (A.M.), dinman@umd.edu (J.D.D.)DOI 10.1016/j.molcel.2007.02.015

SUMMARY

Ribosomal protein L3 (L3) is an essential and in-dispensable component for formation of thepeptidyltransferase center. Atomic resolutionribosome structures reveal two extensions ofL3 protruding deep into the core of the largesubunit. The central extension of L3 in Saccha-romyces cerevisiae was investigated usinga combination of molecular genetic, biochemi-cal, chemical probing, and molecular modelingmethods. A reciprocal relationship betweenribosomal affinity for eEF-1A stimulated bindingof aa-tRNA and for eEF2 suggests that the cen-tral extension of L3 may function as an allostericswitch in coordinating binding of the elongationfactors. Opening of the aa-tRNA accommo-dation corridor promoted resistance to the Asite-specific translational inhibitor anisomycin,suggesting a competitive model for anisomycinresistance. These changes were also found toinhibit peptidyltransferase activity, stimulatingprogrammed �1 ribosomal frameshifting andpromoting virus propagation defects. Thesestudies provide a basis for deeper insight intorational design of small molecule antiviraltherapeutics.

INTRODUCTION

Multicomponent nanoscale biomachines must be able to

coordinate orderly series of functions. For example, the

elongation phase of protein synthesis requires that the ri-

bosome perform at least nine sequentially ordered steps.

The emerging view is one in which each event triggers al-

losteric rearrangements of specific segments of ribosomal

RNAs (rRNAs) and ribosomal proteins, thus enabling infor-

mation transfer among and coordination between the var-

ious functional centers of the ribosome (reviewed in Noller

[2007]). Although rRNA carries out the catalytic activity

and comprises the bulk of the ribosome, many ribosomal

proteins are also required for proper function. Ribosomal

protein L3 (L3) in particular appears to be extremely im-

portant: its sequence and structure are highly conserved

across all three kingdoms (reviewed in Brodersen and

Molec

Nissen [2005]); it is one of the few ribosomal proteins

required for peptidyltransferase activity (Schulze and

Nierhaus, 1982); and it is one of only two proteins capable

of initiating in vitro assembly of E. coli large ribosomal sub-

units (Nowotny and Nierhaus, 1982). Like many ribosomal

proteins, L3 contains a globular domain on the cytoplas-

mic face of the large subunit and has two long tentacle-

like structures that extend deep into the mostly rRNA

core. L3’s central extension reaches all the way to the A

site side of the peptidyltransferase center (PTC), where

a tryptophan at position 255 (in the S. cerevisiae L3 pro-

tein), which is located at its tip, makes the closest ap-

proach of any amino acid to the peptidyltransferase center

active site (Klein et al., 2004; Meskauskas et al., 2005). Re-

gions of two important rRNA helical structures are also

anchored on this central extension (also called the ‘‘W fin-

ger’’ after the tryptophan residue): helix 95, and the struc-

ture formed by helices 90–92. Helix 95 contains the sarcin/

ricin loop (SRL), one of the key structures recognized by

the translation elongation factors, and the target of ribo-

some-inactivating proteins such as ricin (reviewed in Irvin

and Uckun [1992]). The helix 90–92 structure is important

because it forms one side of the corridor along which the

30 ends of aa-tRNAs slide during the process of accom-

modation (Sanbonmatsu et al., 2005). The N-terminal

extension of L3 may also interact with this structure.

Molecular genetic and biochemical studies have shown

that L3 plays an important role in aa-tRNA binding, pepti-

dyltransferase activity, drug resistance, translational

frame maintenance, and virus replication, and that it also

acts as a binding site for a ribosome inhibitory protein

(Wickner et al., 1982; Meskauskas et al., 2003, 2005; Peltz

et al., 1999; Petrov et al., 2004; Bosling et al., 2003; Fried

and Warner, 1981; Hudak et al., 1999; Jimenez et al.,

1975; Pringle et al., 2004; Schindler et al., 1974). Satura-

tion mutagenesis suggested that L3 may function to

transmit information between the SRL and the PTC

(Meskauskas et al., 2005).

The current study employed a combination of targeted

mutagenesis, structural, biochemical, and genetic

approaches to examine the function of the W finger.

Mutagenesis experiments suggest that the tip of this

structure interacts with a nearby base or bases in 25S

rRNA, presumably A2940 (E. coli A2572). The W finger ap-

pears to be intrinsically flexible, but its range of movement

is strictly limited in one direction. Changes in the confor-

mation of the W finger resulting from mutation of W255

to cysteine (W255C) induce significant conformational

ular Cell 25, 877–888, March 23, 2007 ª2007 Elsevier Inc. 877

Molecular Cell

L3: The A Site Gatekeeper

Figure 1. Putative Interaction between

the Tip of the L3 W Finger and A2940 of

25S rRNA Is Critical for Cell Viability

(A) Predicted interactions between amino acid

substitutions at position 255 with large subunit

rRNA. Images were modeled using data for

yeast proteins obtained from cryo-EM studies

threaded into the H. marismortui X-ray crystal

structures (Spahn et al., 2004). U2607 of the

H. marismortui 23S rRNA (shown here) is the

structural equivalent of E. coli A2572, and of

A2940 in the S. cerevisiae 25S rRNA.

(B) Viability of substitution mutants at position

255 of L3 as monitored by 10-fold dilution

spot assays on �Trp 5-FOA plates. Apparently

viable W255E and W255A colonies were

proved to be revertants to tryptophan by DNA

sequence analysis.

rearrangements of the A site side of the PTC, the helix

90–92 structure, helix 95, and the SRL. These are accom-

panied by resistance to anisomycin, increased affinity for

aa-tRNA, decreased affinity for eEF2, and decreased

rates of peptidyltransfer. Other mutants of nearby histi-

dines had the opposite effects on elongation factor and

aa-tRNA binding to ribosomes. These findings suggest

a model in which L3 may play an important role in synchro-

nizing the processes of aa-tRNA accommodation and

translocation by functioning as sensor of the tRNA occu-

pancy status of the A site region of the PTC.

RESULTS

Targeted Substitutions for Tryptophan at Position

255 of L3 Point to a Crucial Interaction with A2940

in the Yeast Ribosome

Previous studies showed that substitution of W255 with

cysteine (W255C) conferred resistance to anisomycin, in-

creased rates of programmed �1 ribosomal frameshifting

(�1 PRF), inability to maintain the killer virus (the Mak�

phenotype), increased affinity for aa-tRNA, and de-

creased peptidyltransferase activity (see Meskauskas

et al. [2003]). This broad spectrum of phenotypes sug-

gested a crucial role for W255 in ribosome function, raising

questions regarding its nature. Molecular modeling using

numerous X-ray crystal structures from H. marismortui

(Ban et al., 2000), E. coli (Schuwirth et al., 2005), and

T. thermophilus (Selmer et al., 2006) reveals that the struc-

ture of this core region of the large subunit is highly

conserved and that W255 is positioned at the tip of the

W finger (data not shown). In the current study, molecular

modeling was based on the H. marismortui structures

because (1) they provided the highest available levels of

resolution, and (2) its amino acid sequence in the central

extension of L3 most closely resembles that of the yeast

protein. This analysis revealed a potential base stacking

interaction along the A site-proximal side of the PTC be-

tween W255 of L3 and A2940 in helix 90 of 25S rRNA

(A2572 in E. coli and U2607 in H. marismortui 23S rRNA,

Figure 1A). The ‘‘plasmid shuffle’’ method (Rose et al.,

878 Molecular Cell 25, 877–888, March 23, 2007 ª2007 Elsevie

1990) was used to determine the viability of a set of amino

acid substitutions for W255 as the only forms of L3. Amino

acid substitutions were chosen to reflect the full range of

biochemical properties of their side chains. In agreement

with the proposed aromatic stacking, substitution of other

aromatic amino acids for tryptophan did not affect cell

growth (Figure 1B, Table 1). Arginine, with its long, basic

side chain, was also viable, but basic substitutions

showed decreased viability in proportion to reductions in

side-chain lengths, indicating that electrostatic interac-

tions may to some extent complement the requirement

for aromatic side chains. The acidic and small amino

acid substitutions, predicted to be unable to interact

with nearby bases (Figure 1A), all resulted in lethal pheno-

types (Figure 1B), as did deletion of W255 (data not

shown). DNA sequence analyses of clones rescued from

apparently viable W255E and W255A colonies revealed

that all were revertants to tryptophan. The set of viable

substitutions clearly indicates the requirement of aromatic

or positively charged amino acids at position 255 for cell

viability, the only exception being the original W255C mu-

tant (see next paragraph addressing this issue). These

findings also point to the importance of putative interac-

tions between the tip of the L3 W finger and rRNA bases,

the most likely candidate being A2940.

Intrinsic but Directionally Constrained Flexibility

of the W Finger

Given that small amino acids at position 255 were lethal,

how could substitution with cysteine be viable? Examina-

tion of nearby amino acids revealed a second cysteine,

C251, located four residues in the N-terminal direction.

We hypothesized that formation of a disulfide bond be-

tween C251 and C255 may promote structural rearrange-

ments of W finger, repositioning nearby aromatic or basic

residues to establish new contacts with A2940 (E. coli

A2572) or other nearby bases. If true, then disruption of

the disulfide bond should prevent such repositioning,

resulting in lethality. Two complementary experiments

were designed to test this hypothesis. Figure 2A shows

that while the W255C and C251S single mutants were

r Inc.

Molecular Cell

L3: The A Site Gatekeeper

Table 1. Summary of Tryptophan Finger Mutants Examined in This Study

Strain Growtha Resistance to Anisomycinb A Site KDc P Site KD

d eEF2 KDe PT Kobs

f Killerg % �1 PRFh

WT + 1 83 ± 9.1 56 ± 3.1 17.4 ± 1.7 0.26 ± 0.02 K+ 8.8 ± 0.3

W255 Mutants

W255F + 1 K+

W255Y + 1 K+

W255R + 3 19 ± 2.1 27.8 ± 2.6 0.09 ± <0.01 K+

W255K Y 2 41 ± 3.2 39.2 ± 8.7 0.05 ± <0.01 K�

W255H YY 1 113 ± 18.4 39 ± 2.9 0.05 ± <0.01 K� 10.8 ± 0.7

W255C + 3 18 ± 1.9 35.1 ± 9.8 0.05 ± <0.01 K� 14.2 ± 0.4

W255A Inviable

W255E Inviable

W255G Inviable

DW255 Inviable

Mutations N Terminal to W255

iV249i Inviable

DV249 j Inviable

iA250 Inviable

DA250 + 1 K+

DA254 + 1 110 ± 13.6 78 ± 4.5 0.09 ± <0.01 K� 10.7 ± 0.2

Mutations C Terminal to W255

H256A Y 1 138 ± 15.1 12.5 ± 1.5 K+

H256Q Y 2 41 ± 4.8 K+

DA258 Inviable

H259A + 1 218 ± 15.0 8.1 ± 1.5 K+

H259L + 1 K+

DV260 Inviable

iV260 Inviable

W262A YY 1 K+

iV264 Y 1 68 ± 5.1 51 ± 4.4 0.05 ± <0.01 K� 12.1 ± 0.4

DA265 Inviable

Double Mutants

W255C/H256A + 3 K�

W255C/H259A Aniso. Dep. 3

W255C/W262A + 3 K�

DV249/DA265 Inviable

DA250/DV260 YY 1

iV249/i264V Inviable

i250A/i260V Inviable

a Growth: + represents wild-type growth rate; down arrow and double down arrows denote moderate and severe growth defects, respectively.b Resistance to anisomycin, expressed in units loosely corresponding to 10-fold changes where 1 = wild type, 2 = 3- to 30-fold more resistant, and

3 > 30-fold more resistant.c A site KD: dissociation constants of ribosome substrates for A site (Phe-tRNA and Phe-tRNA�GTP). ± denotes standard errors throughout.d P site KD: dissociation constants of ribosomes for P site substrate (Ac-Phe-tRNA). ± denotes standard errors throughout.e eEF2 KD: dissociation constants of ribosomes for eEF2. ± denotes standard errors throughout.f Peptidyltransferase activity expressed as the Kobs (min�1) of the puromycin reaction.g Killer phenotypes: K+ denotes wild-type Killer phenotype; K� indicates loss of Killer.h % �1 PRF: efficiency of �1 ribosomal frameshifting.i The letter ‘‘i’’ is used to indicate insertion of an alanine residue at the N-terminal side of the indicated amino acid position.j A D symbol is used to indicate deletion of the specified amino acid.

Molecular Cell 25, 877–888, March 23, 2007 ª2007 Elsevier Inc. 879

Molecular Cell

L3: The A Site Gatekeeper

Figure 2. Intrinsic but Limited Flexibility

of the L3 W Finger

(A) Wild-type L3, W255C, and C251S are viable

as the sole forms of L3, but the W255C/C251S

double mutant is not.

(B) The W255C mutant is hypersensitive to the

reducing agent dithiothreitol.

(C) H259 is absolutely required for viability in

combination with W255C, and H256 is partially

required, suggesting that the putative inter-

actions normally made between W255 and

A2940 (E. coli A2572) are compensated for by

a combination of H259 and H256 in the

W255C mutant.

(D) Insertions or deletions in the W finger,

resulting in shifting of W255 toward the

N-terminal side of the central extension (green

up arrow) are mostly viable (except iV260 and

DV249), while those that shift W255 toward

the C-terminal side (red down arrow) are lethal.

(E) Model describing these observations.

Modeling was based on the cryo-EM map of

the yeast 80S ribosome threaded onto the

X-ray crystal structure of H. marismortui (Spahn

et al., 2004). (Top panel) Mutated cysteine at

position 255 presumably forms a disulfide

bridge with C251 (red dots), repositioning

H256 and H259 to interact with A2940 (shown

as H. marismortui U2607). (Bottom panel) In

general, mutants that allow movement of the

tip of the W finger toward the N-terminal side

of the L3 W finger (green arrow) reposition his-

tidines on the C-terminal side to interact with

A2940, thus helping to restabilize the A site re-

gion of the PTC. In contrast, mutations that

move the tip in the other direction (red arrow)

are lethal because there are no basic or aro-

matic amino acid residues on the N-terminal

side of W255 to interact with A2940.

viable, the W255C/C251S double mutant was lethal.

Figure 2B shows that 4 mM DTT severely inhibited growth

of the W255C mutant, but not of wild-type cells. These re-

sults support the existence of a disulfide bond between

C255-C251 in the W255C mutant.

880 Molecular Cell 25, 877–888, March 23, 2007 ª2007 Elsevie

Formation of a disulfide bond between C255 and C251

would shift the tip of central extension toward the N termi-

nus, potentially moving basic or aromatic residues on the

C-terminal side of C255 into position to interact with

A2940. Examination of the proximal amino acids on the

r Inc.

Molecular Cell

L3: The A Site Gatekeeper

C-terminal side of position 255 revealed three candidates:

H256, H259, and W262. To test this hypothesis, these res-

idues were changed to alanine either individually or in

combination with the W255C mutation (Figure 2C). Among

the single mutants, H256A and H259A did not affect

cell growth, while W262A was viable, albeit it negatively

impacted growth rates. Among the double mutants, the

W255C/H256A allele was viable but conferred reduced

growth, the W255C/H259A double mutant was inviable,

and growth of the W255C/W262A mutant was indistin-

guishable from wild-type cells. These data suggest that

in the W255C mutant, H259 (likely in combination with

H256) is repositioned to interact with A2940, although

the possibility that other bases may be involved cannot

be excluded. A cartoon of this model is shown in the

upper panel of Figure 2E.

These results indicate that the requirement for aromatic

or positively charged residues at the tip of the L3 W finger

can be met by adjacent positive residues, provided that

they are moved into correct position subsequent to for-

mation of the putative disulfide bond between C251 and

C255 in the W255C mutant. Although the data do not allow

determination of the exact conformation of the W finger

in the W255C mutant, they suggest that a certain degree

of flexibility of the W finger in the direction of the N termi-

nus can be tolerated and that this flexibility may be an

inherent feature of L3. The next issue was to determine

the extent and limitations of the W finger’s flexibility. The

amino acid sequence surrounding the tip of the W finger

is N-RKVACIGA(W255)HPAHVMW-C. On the N-terminal

side, the amino acids proximal to W255 are all aliphatic,

and the nearest best candidates for interactions with

rRNA are seven and eight residues away (arginine and

lysine). As discussed above, the C-terminal side contains

the two nearby histidines and a tryptophan. Insertional

mutagenesis of amino acids on the N-terminal side of

W255 would move adjacent N-terminal residues toward

the position occupied by W255, and these mutants should

be inviable. Deletions of residues C terminal to W255

should have similar effects. In contrast, deletions on the

N-terminal side and insertions on the C-terminal side of

W255 were predicted to be viable because they should

bend the tip of the W finger toward the N terminus, moving

H256 into position where it could interact with A2940. To

test this model, L3 mutants containing amino acid dele-

tions or alanine insertions on either side of the W finger

were generated so as to either push or pull the W finger

in the N- or C-terminal directions as described above.

The results of this experiment are shown in Figure 2D.

As predicted, changes which should bend the W finger to-

ward its C-terminal side (indicated by the red down arrow),

e.g., insertions of alanine after V249 and A250, or deleting

A258, V260, and A265, were all lethal. Conversely, three

of the changes predicted to bend the W finger toward its

N-terminal side (indicated by the green arrow), i.e., inser-

tion of alanine after V264, and deleting A250 or A254,

were viable. The lethality due to insertion of alanine after

V260 or by deletion of V249 is likely to have resulted

Mole

from complex and unpredictable changes, either in the lo-

cal ribosome structure or in L3 itself. The effects of sym-

metrical double insertions and deletion mutations in the

W finger, predicted to move the tip of the W finger further

into or away from the PTC, respectively, were also as-

sayed. As shown in Table 1, the symmetrical double inser-

tion mutants (iV249/iV264, iV260/iA250) and one of the

double deletion mutants (DV249/DA265) were lethal. The

DV260/DA250 double deletion mutation was viable but

conferred a very severe growth defect on cells (Table 1).

These findings suggest that the W finger cannot move in

toward or away from the PTC without affecting cell viabil-

ity. In summary, three different series of targeted muta-

tions to the W finger of L3 are consistent with the notion

that the W finger is flexible but that its range of motion is

very tightly constrained within the densely crowded inner

core of the large subunit. Specifically, the data suggest

that its flexibility appears to be limited toward the N-termi-

nal side of W255 in the direction of the extended helix

formed by helices 90–92. A cartoon of this model is shown

in the lower panel of Figure 2E.

Mutations in the W Finger Affect 25S rRNA

Structure in the Sarcin/Ricin Loop, along the

Pathway Taken by Accommodating aa-tRNA,

and the A Site-Proximal Side of the PTC

The data presented above suggested that the W finger

mutants may affect local ribosome structure. To examine

this, ribosomes purified from wild-type and mutant cells

were probed for structural changes using three base-

specific solvent-accessible reagents: dimethylsulfate

(DMS), kethoxal, and carbodiimide metho-p-toluenesulfo-

nate (CMCT). rRNAs were extracted, and modified bases

were identified by primer extension using reverse tran-

scriptase to detect methylation at the N3 position of

uridines and the N1 position of guanosines (CMCT), at

the N1 and N2 positions of guanosines (kethoxal), and at

the N1 position of adenosines and N3 position of cytidines

(DMS) (Inoue et al., 1985). The primers used (see Experi-

mental Procedures) were designed to probe functional

regions of domain V (including the A site and P site loops,

the PTC, and the helices adjacent to these structures, i.e.,

helices 73, 74, 89–93), and helices 94–96.

The results of these experiments are shown in Figures

3A–3C and are summarized in Figures 3D and 3E. The

W255C mutant significantly altered the structure of the

PTC, especially in the A site loop and along helices

90–92 (Figure 3A). In the helix 90–92 structure, A2889,

C2898, G2913, C2924, A2929, A2932, A2935, A2940,

and C2941 were significantly deprotected from chemical

modification. In helix 89, U2861 showed decreased reac-

tivity to CMCT. Similar, albeit weaker deprotection pat-

terns were noted in the W255R, W255H, W255K,

H256Q, and DA254 mutants (data not shown). The stron-

ger effects due to the W255C mutation may reflect the

large conformational changes of the W finger due to the

putative C251-C255 disulfide bond described above.

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Molecular Cell

L3: The A Site Gatekeeper

Figure 3. Structure Probing of Wild-Type and Mutant Ribosomes

(A–C) Autoradiograms of reverse transcriptase primer extension reactions spanning sequence in helices 89–93 (A), helix 73 (B), and the helix 94–95

regions (C). Sequencing reactions (left sides of panels) are labeled corresponding to the rRNA sense strand. Mutants are indicated at top, and bases

with altered chemical protection patterns are indicated to the left. Below each panel, ‘‘U’’ stands for untreated, ‘‘D’’ is DMS, ‘‘C’’ denotes CMCT, and

‘‘K’’ indicates kethoxal.

882 Molecular Cell 25, 877–888, March 23, 2007 ª2007 Elsevier Inc.

Molecular Cell

L3: The A Site Gatekeeper

L3 also makes contacts with bases in helix 73 at the A

site-proximal side of the PTC (Klein et al., 2004). Chemical

protection experiments revealed increased modification

of two bases, A2398 and A2400, in the W255R and

W255K mutants (Figure 3B). The region comprising the

SRL, which forms part of the elongation factor-binding

site, was also probed for structural changes (Figure 3C).

A3020 and A3023, both located in the SRL, and A3010

(base of helix 95) were deprotected in the W255C mutant.

A few additional changes were also noted in the vicinity

of the SRL in the W255R (deprotection of C3003, G3014,

and G3014), H256Q (deprotection of A3023), and DA245

(enhanced protection of A3020) mutants. The major

changes in protection patterns in the vicinity of the PTC

and SRL are summarized on a two-dimensional rendering

of the yeast 25S rRNA (Figure 3D). Molecular modeling of

these changes to the three-dimensional structure of the

H. marismortui large ribosomal subunit (Figure 3E) reveals

that the W finger mutants affect the conformation of re-

gions of 25S rRNA that are involved in interactions with

the aa-tRNA from where it first encounters the ribosome

in complex with eEF-1A, along the pathway taken by

accommodating aa-tRNA (indicated by the orange arrow)

(Sanbonmatsu et al., 2005), and into the A site region of

the PTC in the core of the large subunit. Taken together,

the rRNA chemical protection studies indicate that this

group of W finger mutants promoted a more accessible or

‘‘open’’ conformation of these regions of the large subunit.

An Inverse Relationship between Ribosomal

Affinity for aa-tRNA and eEF2

We previously showed that the W255C mutant promoted

increased affinity for aa-tRNA (Meskauskas et al., 2005).

This analysis was extended in the current study in which

aa-tRNA binding properties were compared between

wild-type and nine mutants. Preparations of non-salt-

washed ribosomes containing soluble factors were used

to monitor aa-tRNA/ribosome interactions because bind-

ing of aa-tRNA to the ribosomal A site requires elongation

factor 1 (Schilling-Bartetzko et al., 1992). The saturation

curves of [14C]Phe-tRNA binding shown in Figure 4A

were analyzed using GraphPad Prism software fitted to a

nonlinear regression one-site binding curve [Y = Bmax*X /

(KD + X)], where Y denotes bound [14C]Phe-tRNA values,

X is input [14C]Phe-tRNA, and Bmax is maximal ligand bind-

ing. As summarized in Table 1, KD values for aa-tRNA var-

ied over an�10-fold range, from a low of�20 nM (W255C

and W255R) to >200 nM (H259A).

Coordination of the translation elongation cycle requires

that binding of the eEF-1A�aa-tRNA�GTP ternary com-

Molec

plex and eEF2 to the elongation factor-binding region be

mutually exclusive. To examine eEF2/ribosome interac-

tions, affinity-purified 63 His-tagged eEF2 was incubated

with GDPNP and salt-washed ribosomes isolated from

cells expressing wild-type L3 as well as six mutant forms

of L3. Ribosomes were harvested by centrifugation, the

pellets were treated with EDTA, and cofractionating

eEF2 was ADP ribosylated using diphtheria toxin and

[14C]NAD+. Ribosome-associated eEF2 was then quanti-

fied by liquid scintillation. Analyses of the saturation

curves (Figure 4B) revealed notable differences in eEF2

binding among the different ribosomes. As summarized

in Table 1, KD values for eEF2 varied over a 5-fold range,

from a low of�8 nM (H259A) to �40 nM (W255K). Inspec-

tion of these data clearly shows a reciprocal relationship

between eEF-1A stimulated binding of aa-tRNA and bind-

ing of eEF2.

Ribosomes isolated from wild-type cells and from three

mutants (DA254, W255H, and iV264) were also assayed

with respect to their affinities for Ac[14C]Phe-tRNA. As

summarized in Table 1 (data from saturation curves shown

in Figure 4C), only small changes in P site binding were ob-

served for the W255H and DA254 mutants. These findings

are consistent with previous data showing that the W255C

mutation did not affect Ac-Phe-tRNA binding to the P site

(Meskauskas et al., 2005).

Anisomycin Resistance Correlates with Increased

Affinity for aa-tRNA

As shown in Figure 3E, anisomycin competes with the 30

end of aa-tRNA for the same binding site in the A site of

the peptidyltransferase center (Hansen et al., 2003). Prior

examination of the randomly generated series of L3 mu-

tants demonstrated a correlation between anisomycin re-

sistance and increased ribosomal affinity for aa-tRNA

(Meskauskas et al., 2005). To further examine this relation-

ship, 10-fold dilution spot assays were performed on rich

media alone or on media containing 50 mg/ml of anisomy-

cin, and growth of cells in the presence of drug relative to

no drug was used to score for anisomycin resistance. The

results of these experiments are summarized in Table 1. At

position 255, the aromatic substitutions (W255F and

W255Y) did not affect anisomycin sensitivity. The longer

basic substitutions (W255R and W255K) showed aniso-

mycin resistance. Changing H256 to alanine (H256A) did

not promote resistance to anisomycin, but substitution

with glutamine (H256Q) promoted drug resistance. Nei-

ther the H259L nor H259A mutants were anisomycin resis-

tant, but these mutations in combination with W255C

(H256A/W255C, W255C/H259A, and W262A/W255C)

(D) Localization of bases in yeast 25S rRNA whose modification patterns were affected by the L3 mutants. Protected and deprotected bases are in-

dicated by open and filled arrows, respectively; bases of 25S rRNA located within 4 A of W255 are boxed in gray.

(E) rRNA protection data mapped onto the H. marismortui large subunit crystal structure. Base numbering follows the S. cerevisiae sequence shown in

(D), and E. coli numbering is shown in parentheses. The mutants described above result in conformational changes of helices 90–92 (orange and

yellow), helix 95 (light blue), and helix 73 (gray). Nucleotides with altered chemical protection patterns are shown in pale blue. Bases forming the

two A site gates are in yellow. Bases interacting with anisomycin are in salmon. A2819 (E. coli A2451) is shown in red. The path taken by the 30

end of the aa-tRNA from the SRL to the PTC (Sanbonmatsu et al., 2005) is shown as an orange arrow.

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L3: The A Site Gatekeeper

Figure 4. Biochemical Characterization of Ribosomes Extracted from Cells Expressing L3 W Finger Mutants

(A) Single site isotherms of eEF-1A stimulated binding of [14C]Phe-tRNA to ribosomal A sites.

(B) eEF2 binding isotherms for wild-type and mutant ribosomes.

(C) Binding isotherms of Ac-[14C]Phe-tRNA to ribosomal P sites.

(D) Characterization of peptidyltransferase activities of wild-type and mutant ribosomes by first order time plots of Ac-[14C]Phe-puromycin formation.

Error bars denote standard deviations.

were all drug resistant. In fact, W255C/H259A, which was

inviable on drug-free media, grew well in the presence of

anisomycin, i.e., it was anisomycin dependent. None of

the viable insertion or deletion mutants affected growth

in the presence of anisomycin. Examination of Table 1

shows that decreasing dissociation constants for

[14C]Phe-tRNA correlated with the degree of anisomycin

resistance. Approximately 4-fold decreases in KD values

correlated with >30-fold increases, and �2-fold de-

creases correlated with 3- to 30-fold increases in aniso-

mycin resistance, respectively.

Increased Rates of�1 PRF and Inability to Propagate

the Killer Virus Correlate with Decreased

Peptidyltransferase Activity

Maintenance of the yeast killer virus is exquisitely sensitive

to subtle defects in the translational apparatus (reviewed

in Wickner [1996]), providing a rapid and sensitive assay

to monitor the effects of the W finger mutants on ribosome

function. The killer virus maintenance phenotypes of the

mutants generated in this study are summarized in Table

1. At position 255, the healthy substitutions (W255F,

W255Y, and W255R) were able to maintain the killer virus

(Mak+ phenotype), but the less viable W255K and W255H

were not (Mak�). In the C-terminal direction, changes at

position 256 (H256A and H256Q) and 259 (H259L and

H259A) did not affect killer virus maintenance. However,

884 Molecular Cell 25, 877–888, March 23, 2007 ª2007 Elsevier

the double mutants of this series in combination with

W255C revealed interesting trends: W255C/H256A and

W262A/W255C were Mak�, indicating that the W255C

mutation is dominant in this aspect. Of the viable insertion

and deletion mutants, two promoted the Mak� phenotype

(DA254 and iV264), and one maintained the killer virus

(DA250). The L-A viral replicase is synthesized by a �1

PRF event; viral particle assembly and propagation of

the killer virus depends on proper rates of�1 PRF (Dinman

and Wickner, 1992). PRF efficiency was monitored for

wild-type cells and four of the Mak� strains (W255H,

W255C, DA254, and iV264). Consistent with previous

studies, there was a strong correlation between increased

levels of �1 PRF and the Mak� phenotype (see Table 1).

Prior studies suggested a correlation between de-

creased peptidyltransferase activity and increased �1

PRF and the Mak� phenotype (Meskauskas et al., 2003),

but a later investigation suggested that other factors

may influence the relationships between these parame-

ters (Meskauskas et al., 2005). Peptidyltransferase activi-

ties were significantly decreased in all six mutants tested

(DA254, W255C, W255H, W255K, W255R, and iV264,

see Figure 4D and Table 1), and in general, this cor-

responded with the inability of cells to maintain the killer

virus (see Table 1). The W255R mutant was the exception:

although peptidyltransferase activity in this mutant was in-

hibited to the same extent as, for example, DA254 (which

Inc.

Molecular Cell

L3: The A Site Gatekeeper

had elevated levels of�1 PRF and was Mak�), this mutant

nonetheless stably maintained the Killer+ phenotype.

DISCUSSION

The elongation cycle requires coordination of an ordered

series of events. The elongation factor 1�aa-tRNA�GTP

ternary complex must first interact with the ribosomes’

factor-binding site. At the decoding center in the small

subunit, the presence of cognate codon:anticodon

base-pairing is transduced up the body of the aa-tRNA,

activating GTP hydrolysis by elongation factor 1 (Valle

et al., 2002; Cochella and Green, 2005). This initiates ac-

commodation of the aa-tRNA into the PTC. Successful ac-

commodation has been hypothesized to be accompanied

by rRNA structural changes that both close the aa-tRNA

accommodation corridor (Sanbonmatsu et al., 2005),

and activate the PTC (Schmeing et al., 2005). Concur-

rently, the structure of the factor-binding site must also

change to favor elongation factor 2 binding, setting the

stage for translocation (Sergiev et al., 2005). The rRNA

structure probing and ribosome binding data presented

in the current study suggest a model describing how L3

may help to coordinate these ribosome-associated func-

tions by acting as a ‘‘gatekeeper’’ to the A site.

Computer modeling studies suggest that during ac-

commodation, the aa-tRNA 30 end moves through a ‘‘cor-

ridor’’ formed by helix 89 on one side and the helix 90–92

structure on the other (Sanbonmatsu et al., 2005). The

chemical protection data clearly show that specific bases

in the helix 90–92 side of this pathway are generally depro-

tected in the W255C mutant as compared to wild-type ri-

bosomes. Thus, the increased accessibility of bases in

this structure to small chemicals suggests that it is in a rel-

atively open conformation (Figure 3A). The computational

simulations of aa-tRNA accommodation also suggest that

the aa-tRNA 30 end passes through two rRNA-based

structures or ‘‘gates.’’ The first gate encountered by aa-

tRNA (gate 1) is formed by U2491 and C2556 (yeast

U2860 and C2924), while the second (gate 2) is formed

by C2573 (yeast C2941) (see Figures 3D and 3E). Notably,

the gate bases in the helix 90–92 structure (C2924 and

C2941) were deprotected, consistent with an open con-

formation. Interestingly, U2861 was protected from chem-

ical attack. It is possible that reorientation of the helix 89

gate 1 base U2860 had a shielding effect on U2861, con-

sistent with the idea that the W255C mutant promotes

opening of the accommodation corridor. Additionally,

the chemical protection studies suggest that these con-

formational changes in this and other mutants are promul-

gated along the entire pathway traveled by the aa-tRNA 30

end, from the SRL down to the PTC (Figures 3B and 3C).

Opening of the aa-tRNA accommodation corridor

should affect the biochemical properties of the ribosome.

Specifically, such changes should promote increased af-

finity for aa-tRNA, which is supported by the observation

of decreased KD values for aa-tRNA by the W255C,

W255R, W255K, and H256Q mutants. The increased

Molec

eEF2 KD values for these mutants are also consistent

with the notion that ternary complex and eEF2 binding

should be mutually exclusive. This is further supported

by the H256A and H259A mutants, which promoted in-

creased affinity for eEF2 at the expense of their ability to

interact with aa-tRNA. These biochemical observations

suggest that two classes of mutants were generated:

one promoting ribosomal conformations more favorable

to eEF2 binding, the other favoring binding of the eEF-

1A�aa-tRNA�GTP ternary complex. These data suggest

that in the L3 mutants, bases in the PTC and along the

aa-tRNA accommodation pathway have been artificially

shifted into conformations that are normally transitory dur-

ing translation in wild-type cells. Specifically, we propose

that some W finger mutants (e.g., W255C, W255R,

W255K, and perhaps H256Q) favor positioning of the

SRL into the eEF-1A�aa-tRNA�GTP binding state, and

the accommodation corridor into the open conformation,

while others (H256A and H269A) favor the closure of the

corridor and eEF2 binding.

These findings suggest a model wherein the W finger

may normally assume different positions within the ribo-

some during these two factor-binding states, possibly ac-

companied by different patterns of putative interactions

between amino acids in the W finger and 25S rRNA bases.

This is cartooned in Figure 5. By this model, movement of

the L3 W finger may participate, either actively or pas-

sively, in an allosteric process to coordinate these events.

Specifically, positioning of the W finger toward its N-termi-

nal side, i.e., directed away from the PTC and toward the

factor-binding site, may help both to open the helix 90–92

face of the accommodation corridor and to position the

SRL so as to favor binding of the eEF-1A�aa-tRNA�GTP

ternary complex. We suggest that, while in this conforma-

tion, A2940 of 25S rRNA may interact with H259 of L3. The

model proposes that, during accommodation, the W fin-

ger swings toward its C-terminal side (toward the PTC)

and that the A2940-W255 interaction is established upon

completion of aa-tRNA accommodation into the A site.

Additionally, because the aa-tRNA and eEF2 KD values

for the H256A mutant were between those observed for

the W255 mutants (W255C, W255R, and W255K) and

H259A, it is also possible that interaction between

A2940 and H256 could constitute a transitional inter-

mediate between the two states. We propose that, during

rearrangement of the W finger, the helix 90–92 structure

moves to close the accommodation gates. Concurrently,

the SRL atop helix 95 is repositioned so as to form the

eEF2 competent binding site. This sequence of events

would allow the occupancy status of the A site to be com-

municated to the translation factor-binding region, pre-

venting entry of new aa-tRNA into the A site during trans-

location. It must be noted that our results have only

revealed amino acid residues in the W finger that may be

involved in these conformational events, not their rRNA

counterpart bases. Although the chemical properties of

the substituted amino acids suggest their involvement

in interactions with nucleotides, the actual rRNA base

ular Cell 25, 877–888, March 23, 2007 ª2007 Elsevier Inc. 885

Molecular Cell

L3: The A Site Gatekeeper

Figure 5. Model for the Role of L3 in

Coordinating Functions of the Elongation

Factor-Binding Site: L3 as the Gate-

keeper to the A Site

(Left) Positioning of the L3 W finger (green) away

from the PTC (red oval) positions the helix 90–

92 side of the aa-tRNA accommodation corri-

dor so as to open the accommodation gates

(red circles), allowing accommodation (dotted

gray line) of the 30 end of aa-tRNA (aa). ‘‘pep’’

denotes the 30 end of the peptidyl-tRNA. This

W finger conformation also positions helix 95

to form the eEF-1A�aa-tRNA�GTP ternary

complex binding site and promotes interaction between L3 H259 and 25S rRNA A2940, denoted by ‘‘A’’ (E. coli A2572). (Right) During/after accom-

modation, repositioning of the W finger toward the PTC closes the gates, repositions helices 95 and 90–92 to form the eEF2 binding site, and

promotes the W255-A2940 stacking interaction.

involved was not experimentally determined, but A2940

represents the most likely candidate based on the crystal

structures.

Resistance of the W255C mutant to anisomycin has

been known for many years (Fried and Warner, 1981),

but the underlying mechanism has been unclear. Early

studies showed that the W255C mutation did not affect

anisomycin binding (Jimenez and Vazquez, 1975), sug-

gesting that structural and/or conformational changes in

resistant ribosomes may reduce the ability of anisomycin

to compete with aa-tRNA for entry into the A site. The

chemical protection data presented in the current study

show structural changes in the large subunit consistent

with a more open conformation in the vicinity of the A

site of the PTC and in the aa-tRNA accommodation corri-

dor in the anisomycin-resistant mutants. Opening of the

accommodation corridor should facilitate aa-tRNA pas-

sage into the A site, consistent with the decreased KD

values for aa-tRNAs. In contrast, because of its small mo-

lecular radius relative to aa-tRNA, access to the PTC by

anisomycin should not be as intrinsically limited by the

two gates, and hence should not be stimulated as much

by gate opening. Therefore, it is possible that these muta-

tions lower the barriers encountered by aa-tRNA relative

to anisomycin during accommodation, thus altering the ki-

netic partitioning ratios for these two molecules, resulting

in anisomycin resistance. An alternate, nonexclusive ex-

planation may be that changes in the interactions between

anisomycin and bases in the PTC (see Figure 3E) conse-

quent to these mutations may contribute to anisomycin re-

sistance. Interestingly, the �OH group of the N-terminal

serine of L3 (S2) is predicted to interact with C2924, the

helix 90 ‘‘Gate 1’’ residue (see Figure 3E). The S2T mutant

was also strongly anisomycin resistant (Meskauskas et al.,

2005), and experiments are ongoing to characterize

a bank of S2 mutants.

An ‘‘entropic catalysis’’ model of peptidyltransfer posits

that precise positioning of substrates and water reorgani-

zation in the PTC allows formation of a highly ordered

electrostatic network that stabilizes reaction intermedi-

ates, providing the proper environment for the ribosome

to accelerate peptide bond formation (reviewed in Rod-

nina et al. [2007]). The rRNA structure-probing experi-

886 Molecular Cell 25, 877–888, March 23, 2007 ª2007 Elsevie

ments in the current study are consistent with this model

insofar as distortion of the topology of the A site side of

the PTC by the W finger mutants could affect positioning

of the aa-tRNA 30 end, promoting the observed peptidyl-

transfer defects. It has also been proposed that the PTC

is activated by an ‘‘induced fit’’ mechanism, wherein ac-

commodation of the aa-tRNA acceptor stem into the A

site reorganizes the geometry of the PTC, making the ester

group of the peptidyl-tRNA accessible for nucleophilic at-

tack (Schmeing et al., 2005). The involvement of the L3 W

finger in this process remains an intriguing possibility.

It is remarkable how atomic scale allosteric events can

propagate themselves all the way to the biological level.

Translating our understanding of molecular structure and

biochemical properties of molecules may lead to concrete

medical benefits. For example, understanding how these

mutants affect the interactions between ribosomes and

trans-acting factors and how this leads to resistance to

A site-specific antibiotics can provide the foundation for

the design of new antibacterials to counter resistance of

pathogenic bacteria to drugs such as tiamulin (Pringle

et al., 2004). This research may also aid in the develop-

ment of new antivirals insofar as it furthers our under-

standing of how changes at the atomic level can affect

peptidyltransfer, and hence �1 PRF and virus propaga-

tion. It is hoped that atomic resolution structures of yeast

ribosomes will be solved in the near future and that

achievement of this milestone will enable the models pro-

posed herein to be directly visualized.

EXPERIMENTAL PROCEDURES

Strains, Plasmids, Genetic Manipulation, and Media

E. coli DH5a was used to amplify plasmid DNA. Transformation of

E. coli and yeast, and preparation of yeast growth media (YPAD,

synthetic drop out medium, and 4.7 MB plates for testing the Killer

phenotype), were as previously reported (Dinman and Wickner,

1992). Restriction enzymes were obtained from MBI Fermentas

(Vilnius, Lithuania). The QuikChange XL II Site-Directed Specific Muta-

genesis Kit was obtained from Stratagene (La Jolla, CA). Macrogen

(Seoul, South Korea) performed DNA sequence analysis. Oligonucleo-

tide primers were purchased from IDT (Coralville, IA). The yeast strains

used in this study were all derived from the rpl3-gene disruption (rpl3D)

strain JD1090 (MATa ura3-52 lys2-801 trp1d leu2- his3 RPL3::HIS3

pRPL3-URA3-CEN6 [L-A HN M1]) (Meskauskas et al., 2003). Mutants

r Inc.

Molecular Cell

L3: The A Site Gatekeeper

of rpl3 were generated using the wild-type RPL3 gene in pJD225

(Meskauskas et al., 2003), synthetic oligonucleotides, and the Quik-

Change XL II Kit. The pYDL dual luciferase reporter series of plasmids

for monitoring programmed ribosomal frameshifting (PRF) were used

as previously described (Harger and Dinman, 2003). Ten-fold dilution

spot assays were performed on rich media alone and on media con-

taining 50 mg/ml of anisomycin, and growth of cells in the presence

of drug relative to no drug was used to score for anisomycin

resistance.

Characterization of Peptidyltransferase Activity, tRNA,

and eEF2 Binding Studies

Yeast phenylalanyl-tRNAs were aminoacylated with [14C]Phe-tRNA to

monitor A site binding, Ac-[14C]Phe-tRNA was generated to monitor P

site binding, these were purified by HPLC, and equilibrium binding

studies were performed as previously described (Meskauskas et al.,

2005). Peptidyltransfer assays were performed essentially as previ-

ously described (Dresios et al., 2001; Meskauskas et al., 2005). 63

His-tagged eEF2 was purified from TKY675 yeast cells (kindly pro-

vided by Dr. T. Kinzy) as previously described (Ortiz et al., 2006). For

eEF2 binding experiments, reaction mixes containing 25 pmol of

salt-washed 80S ribosomes and various concentrations of 63 His-

tagged eEF2 (5–40 pmol) in binding buffer (50 mM Tris-HCl [pH 7.5],

50 mM ammonium acetate, 10 mM magnesium acetate, 2 mM DTT,

and 100 mM GDPNP) were incubated for 5 min at room temperature,

layered on top of the 300 ml sucrose cushion (10% sucrose in binding

buffer), and centrifuged at 50,000 rpm for 20 min at 4�C in a MLS50

(Beckman) rotor. Pellets (bound fraction) were washed and resus-

pended in 50 ml of DT buffer (50 mM Tris-HCl [pH 7.5], 20 mM DTT,

and 1 mM EDTA). Ribosome-bound eEF2 was ADP ribosylated for

20 min at 37�C by adding 100 pmol of [14C]NAD+ and 0.2 mg of diph-

theria toxin. Reaction mixes were precipitated with TCA, and amounts

of [14C]ADP-ribosylated eEF2 were determined by liquid scintillation

counting. Control values (lacking eEF2, not exceeding 10% of the low-

est count) were subtracted. KD values were determined assuming sin-

gle binding sites using GraphPad Prism software.

Isolation and Chemical Probing of Mutant Ribosomes

Ribosomes were isolated as described (Meskauskas et al., 2005) and

were synchronized by puromycin treatment in buffer C (50 mM Tris-

HCl [pH 7.5], 5 mM magnesium acetate, 50 mM ammonium chloride,

0.1 mM PMSF, 0.1 mM DTE, and 25% glycerol) containing 1 mM

GTP, 1 mM puromycin, and 500 pmol of ribosomes. After incubation

for 30 min at 30�C, ribosomes were sedimented through a cushion

of buffer B (20 mM Tris-HCl [pH 7.5], 10 mM magnesium acetate,

0.1 mM PMSF, 0.1 mM DTE, 0.5 M KCl, and 25% glycerol) and

suspended in buffer C at 10 pmol/ml. Chemical probing with DMS,

kethoxal, and CMCT, followed by RT primer extension analysis of

modified RNAs, was performed as described (Stern et al., 1988).

Primers (numbered after the first transcribed base of yeast 25S

rRNA) employed for these analyses were 2957 (50-AACCTGTCTCAC

GACGG-30), 3043 (50-CCTGATCAGACAGCCGC-30), 2877 (50-GGTAT

GATAGGAAGAGC-30), 2435 (50-CCTCTATGTCTCTTCAC-30 ), and 2675

(50-GTTCTACTGGAGATTTCTG-30).

Computational Analysis of Ribosome Structure

The X-ray crystal structure of the H. marismortui 50S ribosomal subunit

(Ban et al., 2000) and the cryoelectron microscopy (cryo-EM) recon-

struction of Saccharomyces cerevisiae ribosomal proteins threaded

onto the X-ray crystal structure of the H. marismortui 50S ribosomal

subunit (Spahn et al., 2004) were visualized using PyMOL (DeLano

Scientific LLC).

ACKNOWLEDGMENTS

We would like to thank the members of our laboratory, with special

thanks to Jennifer Baxter-Roshek and Alexey Petrov for stimulating

Mole

discussions, and to Rasa Rakauskaite and Johnathan Russ for techni-

cal assistance. This work was supported by grants from the NIH to

J.D.D. (GM58859) and from the American Heart Association to A.M.

(AHA 0630163N).

Received: November 27, 2006

Revised: January 22, 2007

Accepted: February 14, 2007

Published: March 22, 2007

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Ribosomal protein L3 is involved in replication or maintenance of the

killer double-stranded RNA genome of Saccharomyces cerevisiae.

Proc. Natl. Acad. Sci. USA 79, 4706–4708.

r Inc.