Decoding fidelity at the ribosomal A and P sites: influence of mutations in
three different regions of the decoding domain in 16S rRNA
Decoding fidelity at the ribosomal A and P sites: influence of mutations in three different regions of the decoding domain in 16S rRNA
Michael
O'Connor*
,
Cheryl L.
Thomas
1
,
Robert A.
Zimmermann
1
and
Albert E.
Dahlberg
Department of Molecular and Cell Biology and Biochemistry, Box G, J.W.Wilson
Laboratory, Brown University,
Providence
, RI 02912,
USA
and
1
Department of Biochemistry and Molecular Biology and Program in Molecular and
Cellular Biology, University of Massachusetts,
Amherst
, MA 01003,
USA
Received December 2, 1996;
Revised and Accepted January 28, 1997
ABSTRACT
The involvement of defined regions of
Escherichia coli
16S rRNA in the fidelity of decoding has been examined by analyzing the effects of rRNA mutations on misreading errors at the ribosomal A and P sites. Mutations in the 1400-1500 region, the 530 loop and in the 1050/1200 region (helix 34) all
caused readthrough of stop codons and frameshifting during elongation and
stimulated initiation from non-AUG codons at the initiation of protein synthesis. These results indicate
the involvement of all three regions of 16S rRNA in decoding functions at both
the A and P sites. The functional similarity of all three mutant classes are
consistent with close physical proximity of the 1400- 1500 region, the 530 loop and helix 34 and suggest that all three
regions of rRNA comprise a decoding domain in the ribosome.
INTRODUCTION
Accurate decoding of mRNA requires the active participation of ribosomal
components. The involvement of ribosomal proteins in this process was
demonstrated by classical genetic analyses, which implicated several ribosomal
proteins in decoding fidelity (
1
). While no ribosomal RNA mutations were isolated in these studies, evidence
from many different sources has identified rRNA as an indispensable component
in all steps of translation. The sites of interaction of tRNA, mRNA and other
ligands with rRNA have been identified using crosslinking, chemical footprinting and site-directed mutagenesis (reviewed in
2
). Among the first tRNA-ribosome crosslinks to be obtained was a zero length crosslink between
base 34 of the anticodon of P site-bound tRNA and C1400 of 16S rRNA (
3
). Many of the subsequent biochemical and genetic studies have corroborated the importance of the 1400-1500 region of the small subunit rRNA in tRNA and antibiotic binding,
decoding and subunit-subunit interaction. Moreover, Purohit and Stern (
4
) have shown that a short oligoribonucleotide that mimics the 1400-1500 region of the small subunit rRNA can interact with aminoglycoside
antibiotics and tRNAs in a way that is surprisingly similar to interaction of
the intact 16S rRNA molecule with these ligands.
In the light of the wealth of data linking the 1400-1500 region of 16S rRNA with decoding, this region has been termed the
decoding center or decoding site. However, other regions of the 16S rRNA
molecule also participate in ribosome-tRNA-mRNA interactions (
5
). Furthermore, site-directed mRNA-rRNA crosslinking has shown that as well as the 1400-1500 region, the 530 loop and the 1050/1200 regions of helix
34 (Fig.
1
) are also close to the site of codon-anticodon interaction (
6
). In order to emphasize the contribution of rRNA nucleotides outside the 1400-1500 region to decoding, the term `decoding domain' has been used to
denote all regions of 16S rRNA that impinge upon decoding (
7
).
The functional role of particular sites in rRNA have been analyzed by examining
the effects of mutations at these sites on defined ribosomal functions (
8
,
9
). In the present study, we have analyzed the effects of mutations in three
distinct regions of the decoding domain on tRNA selection during the initiation
and elongation phases of translation. In common with previously characterized
base substitution mutations in the 530 loop and helix 34 (
10
,
11
), we have found that a variety of mutations in the 1400-1500 region of 16S rRNA promote stop codon readthrough and frameshifting
during elongation. This indicates that these mutations influence tRNA selection
at the ribosomal A site. However, mutations in these same three regions of 16S
rRNA, as well as the lack of post-transcriptional modification of bases A1518 and A1519, increase the
frequency of initiation from non-AUG codons, indicating that P site decoding is also affected in these
mutants. Together, our data show that tRNA selection at both the A and P sites
is influenced by the same three regions of 16S rRNA and are consistent with the
notion that these three regions are in close proximity to the site of codon-anticodon interaction in the ribosome.
MATERIALS AND METHODS
Bacterial strains
Strain M41 and its
recA
-
derivative MC140 were used as the standard wild-type strains (
12
). Kasugamycin-resistant (
ksgA
) mutants were obtained from the late Dr Peter van Knippenberg (University of Leiden). The
ksgA19
mutation was transferred into the MC41 genetic background by transducing strain
MC86 (MC41
thr34
::Tn
10
) to threonine independence with phage P1 prepared on the
ksgA19
-containing strain and screening for kasugamycin resistance. The resultant strain was designated MC178.
AUG AUU ACG CUA AGC UUU GUU UAG GCC GGC CCU AAU UCA
CUG
pSG163
UAG mutant
AUG AUU ACG CUA AGC UUA GGG UAU CUU UAG CUA CGG GGC CCU AAU UCA
CUG
pSG627
UAA mutant
AUG AUU ACG CUA AGC UUU GUG GAA UAA GUU AGC GGC CCU AAU UCA
CUG
pSG853
UAA mutant
AUG AUU ACG CUA AGC UUU GUC UAA GUU AGC GGC CCU AAU UCA
CUG
pSG34-11
UGA mutant
AUG AUU ACG CUA AGC UUU GUG UGA GCC GGC CCU AAU UCA
CUG
pSG3/4
UGA mutant
AUG AUU ACG CUA AGC UUA GGG UAU CUU UGA CUA CGA CGG AUC CCC GGG AAU UCA
CUG
pSG12DP
-1 frameshift
AUG AUU ACG CUA AGC UUG GG AUA AGG AUC CCC GGG AAU UCA
CUG
pSGlac7
+1 frameshift
AUG AUU ACG CUA AGC UUU GUGU AGG GUU AGC GGC CCU AAU UCA
CUG
The underlined
CUG
leucine codon corresponds to codon 7 of the wild type lacZ gene.
rRNA and
lacZ
plasmids
Mutant rRNA was expressed from two different sets of plasmids in this study. In
pKK3535, the intact
rrnB
operon is transcribed constitutively from the native P
1
P
2
promoters (
13
). In pNO2680 (
14
), rRNA is transcribed from the [lambda] P
L
promoter. In the presence of the temperature-sensitive [lambda] cI repressor (supplied on the neomycin-resistant, pSC101-derived plasmid pLG857;
12
), transcription of plasmid encoded rRNA is repressed at 30oC but can be induced upon shifting the culture to 42oC. A number of single base substitutions in 16S rRNA, including
C1395U, [Delta]C1400, C1407U, G1505U, C1192U and G529U, were carried on pNO2680-derived plasmids (
15
,
16
). Mutations at positions A792 and G1530/A1531 were carried on pKK3535-derived plasmids, which contained, in addition, the spectinomycin resistance C1192U mutation (
17
,
18
).
The pSG series of plasmids containing frameshift and nonsense mutations in the 5'-end of the
lacZ
gene have been described previously and the relevant sequences are given in Table
1
(
11
,
12
). Plasmid pSG25 is derived from pACYC184 and is compatible with both the pSC101-derived plasmid pLG857 encoding the [lambda] repressor and the pBR322-derived,
rrnB
-containing plasmids pNO2680 and pKK3535. The AUG initiation codon in the wild-type
lacZ
plasmid pSG25 is bounded on the 5'- and 3'-sides by
Eco
RI and
Hin
dIII sites respectively. Plasmids containing non-AUG codons were constructed by replacing the 20 bp
Eco
RI-
Hin
dIII fragment with pairs of complementary synthetic oligonucleotides containing AUN or NUG initiation codons. The primary structure of all mutant plasmids was verified by nucleotide
sequencing and the relevant sequences are listed in Table
1
.
Culture media and growth conditions
Bacteria were routinely cultured in Luria-Bertani (LB) medium. Antibiotics were added as required at the following concentrations: tetracycline, 12.5 [mu]g/ml; neomycin, 50 [mu]g/ml; ampicillin, 250 [mu]g/ml; kasugamycin, 100 [mu]g/ml. [beta]-Galactosidase activities in cells harboring
both mutant rRNA and pSG plasmids were measured after dilution of overnight
cultures and growth at 42oC (for the pNO2680-derived plasmids) or 37oC (for pKK3535 and its derivatives) for 150 min and assayed as previously described
(
12
).
Isolation and sequencing of
[beta]
-galactosidase
To isolate [beta]-galactosidase from strains containing a
lacZ
construct and a pNO2680-derived rRNA plasmid, 2 l LB medium were inoculated with 200 ml cells that
had been grown overnight at 30oC. Expression of the mutant rRNA was induced by growing these cultures at
42oC for 3-5 h. Cells were harvested and [beta]-galactosidase was isolated, purified and sequenced as
described previously (
19
).
RESULTS
Mutations in the decoding center of 16S rRNA decrease the fidelity of A site
decoding
We have previously described the construction of mutations at positions C1395,
C1400, C1407 and G1505. Deletion of C1400 and C -> U mutations at positions 1395 and 1407 had a dominant lethal phenotype and
the mutant rRNAs could only be expressed transiently from an inducible promoter
(
15
). Mutations at G1505 had little effect on cell viability and, when combined
with the C1395U, [Delta]C1400 or C1407U mutations, suppressed lethality of the single base
mutations. As part of our analysis of the influence of defined regions of 16S
rRNA on decoding functions, we have examined the effects of these mutations on
stop codon readthrough and frameshifting. Strains carrying plasmids encoding a
temperature-sensitive allele of the [lambda] cI repressor and
lacZ
constructs containing stop codons or frameshifts in the 5' portion of the coding region were transformed with the wild-type rRNA plasmid (pNO2680) and each of the mutant rRNA plasmids
and transcription of the mutant rRNA was induced by shifting the cultures to 42oC. The data in Table
2
show that mutations at C1400, C1395, C1407 and G1505 caused a 2- to 5-fold increase in the level of readthrough of stop codons and
frameshifting. Readthrough of stop codons occurs by binding of a near-cognate tRNA to the termination triplet in the A site, while frameshifting
can be related to both A and P site decoding events (
20
). These data show that one effect of mutations in the decoding center is to
perturb codon-anticodon interactions in the A site and decrease the fidelity of
elongating ribosomes.
Effects of rRNA mutations on stop codon readthrough and frameshifting
rRNA Mutation
LacZ Plasmids
pSG12-6
pSG163
pSG627
pSG853
pSG34-11
pSG3/4
pSG12DP
pSGlac7
UAG
UAG
UAA
UAA
UGA
UGA
(-1)
(+1)
pNO2680 (wt)
16 +- 2
37 +- 3
5 +- 1
7 +- 1
35 +- 7
107 +- 14
85 +- 4
56 +- 3
pPL133 (C1407U)
29 +- 1
95 +- 3
9 +- 1
44 +- 5
59 +- 5
224 +- 13
241 +- 22
222 +- 6
pPL111 (C1395U)
28 +- 1
61 +- 12
8 +- 1
22 +- 3
58 +- 4
171 +- 30
241 +- 38
165 +- 13
pPLAM5 ([Delta]C1400)
28 +- 2
101 +- 2
10 +- 2
44 +- 4
59 +- 2
214 +- 13
255 +- 31
221 +- 2
pPL1301m26 (G1505U)
24 +- 2
62 +- 3
8 +- 1
23 +- 1
52 +- 2
169 +- 23
142 +- 10
123 +- 9
pPLAM5m26 ([Delta]C1400/G1505U)
30 +- 5
87 +- 3
7 +- 1
24 +- 1
51 +- 4
147 +- 17
256 +- 13
215 +- 13
pPL111m28 (C1395U/G1505U)
29 +- 4
92 +- 3
6 +- 1
28 +- 1
47 +- 6
144 +- 8
257 +- 14
206 +- 11
pPL133m26 (C1407U/G1505U)
26 +- 4
92 +- 2
7 +- 1
30 +- 2
46 +- 5
151 +- 9
235 +- 20
181 +- 11
pNOC1192U
19 +- 1
40 +- 1
5 +- 1
10 +- 1
41 +- 1
135 +- 7
109 +- 3
57 +- 3
Values for stop codon readthrough and frameshifting are expressed in Miller
units of [beta]-galactosidase activity (56). Each value for [beta]-galactosidase activity is the result of 3-5 independent
experiments. Assay conditions are described in Materials and Methods.
Characterization of ribosomal frameshifting events by protein sequencing
The mechanism of frameshifting promoted by the rRNA mutations described in the
preceding section and in our previous work (
11
,
16
) was analyzed further by N-terminal sequencing of [beta]-galactosidase isolated from strains carrying selected rRNA
mutations and
lacZ
frameshift constructs. Protein sequencing of [beta]-galactosidase isolated from a strain expressing the C1407U rRNA
mutation and the pSG12DP -1
lacZ
frameshift gave the following sequence: M I T (L+R) S L G I R I P (Fig.
2
). This suggested that, at the site of frameshifting (UUG GGA UAA), a GGA-decoding tRNA
Gly
slipped backwards by one base onto the 5' overlapping GGG glycine codon. This frameshift site, where a string of
repetitive nucleotides is bordered on the 3'-side by an in-frame stop codon constitutes a `shifty stop' and has been
studied extensively by Weiss
et al.
(
21
). Their analyses have uncovered the importance of an overlapping cognate codon
in the new reading frame and an adjoining stop codon in the original reading
frame. The enhancing effect of the stop codon suggests that tRNA slippage
occurs when the tRNA
Gly
is in the P site and the stop codon is located in the A site. Protein
sequencing showed that at position 4, arginine as well as the expected leucine
were recovered (Fig.
2
). The origin of arginine at this position is unknown, but it may derive from
(mis)reading of the CUG leucine codon by a CGG-decoding tRNA
Arg
via a second position codon-anticodon mismatch.
Effects of decoding domain mutations on codon recognition at the P site
The AUG triplet is overwhelmingly the most common initiation codon in all
organisms, GUG and UUG are infrequently used and AUU, AUA and AUC are rarely,
if ever, used. The initiator tRNA is unique in that it binds directly to the
ribosomal P site. Initiation factors and ribosomal proteins are involved in
selection of the correct mRNA initiation codon and tRNA and actively discriminate against non-initiator tRNAs and codons. In an effort to characterize the effects of rRNA mutations on P site function, we have examined their
influence on the levels of initiation from non-AUG codons. We reasoned that mutations that affected decoding function in
the P site might alter the frequency of initiation from non-AUG initiation triplets. The AUG initiation codon of the wild-type
lacZ
plasmid pSG25 was replaced by GUG, UUG and CUG (pSG431, pSG414 and pSG413 respectively) or AUA, AUC and AUU (pSG416, pSG415 and pSG417 respectively). Decoding of these
triplets by tRNA
f
Met
during initiation from the non-standard initiation codons involves single base codon-anticodon mismatches at the first or third positions, and is thus
analogous to readthrough of UGA codons by tRNA
Trp
or UAA and UAG codons by tRNA
Gln
in the A site (
25
-
27
). The data in Table
3
show that the mutations in the 530 loop (G529U) and helix 34 (U1199C/C1200U and
U1199G/C1200G), as well as the mutations in the C1400 region described in the
preceding section, all stimulated initiation from each of the non-AUG initiation codons. Only some of the helix 34 mutations promoted
miscoding, however. Thus, the C1192U mutation in helix 34, that confers
resistance to spectinomycin (
28
), had no effect on stop codon readthrough, frameshifting or initiation events
(Tables
2
and
3
), consistent with its lack of effect on cell growth or
in vitro
translation parameters (
29
). In order to characterize these aberrant initiation events, [beta]-galactosidase was purified from selected strains and subjected to N-terminal sequencing. Sequence analysis of [beta]-galactosidase isolated from a strain carrying the
pSG413
lacZ
construct and the G529U rRNA mutation showed that, as predicted, initiation
occurred at the CUG codon (Fig.
4
). Similarly, protein sequencing showed that in a strain carrying the pSG415
lacZ
plasmid and the U1199G/C1200G rRNA mutations, initiation occurred at the predicted AUC codon (Fig.
5
). These data show clearly that mutations in three different regions of 16S rRNA
affect codon-anticodon interactions at the ribosomal P site, while the data presented
in the preceding section and in our previous analyses (
10
,
11
,
16
) show that these same regions of rRNA are involved in tRNA selection at the A
site and in reading frame maintenance. The similarity of the functional effects
of these mutants suggests that the 530 loop, the C1400 region and helix 34 are all involved in modulating tRNA-ribosome interactions at multiple steps during translation. Moreover, the isolation of
suppressor/antisuppressor mutations at equivalent positions in yeast
mitochondrial and cytoplasmic small subunit rRNAs (under conditions where all the rRNA is mutant) suggests the involvement of these three regions of rRNA in decoding in all organisms (
7
).
Initiation of translation requires the participation of the three initiation
factors IF1, IF2 and IF3. IF3 is involved in selection of the initiator tRNA
that binds to the P site of the small subunit. The factor interacts with the
anticodon stem-loop region of the tRNA and destabilizes initiation complexes containing
non-initiator tRNAs or non-canonical initiation complexes (
30
). Crosslinking experiments have shown that IF3 interacts with both the central
and 3' minor domains of 16S rRNA (
31
) and 30S ribosomal subunits containing mutations at positions G791 and A792 in
the 790 loop or at G1530/A1531 at the 3'-end of 16S rRNA were shown to be defective in IF3 binding (
17
,
18
,
32
). Because of the influence of these rRNA mutations on ribosome-IF3 interactions, we have analyzed their effects on selection of AUG
initiation codons. These data, presented in Table
3
, show that the A792U, A792G and G1530A/A1531G mutants do not increase
initiation from non-AUG codons. This indicates that decreases in ribosome-IF3 interaction
per se
do not affect the selection of AUG initiation codons, but instead suggests that
the rRNA mutations analyzed here affect initiation by decreasing the accuracy
of tRNA selection in the ribosomal P site.
Effects of rRNA mutations on initiation from non-AUG codons
rRNA plasmid/
LacZ plasmids initiation codons
a
mutation
pSG25-AUG
pSG413-CUG
pSG414-UUG
pSG431-GUG
pSG415-AUC
pSG416-AUA
pSG417-AUU
pNO2680 (wt)
7298 +- 222
115 +- 13
1739 +- 238
3428 +- 39
87 +- 5
63 +- 3
80 +- 5
pPL133 (C1407U)
8592 +- 453
241 +- 45
3707 +- 187
4078 +- 259
333 +- 16
210 +- 4
315 +- 10
pPL111 (C1395U)
9118 +- 445
174 +- 20
3165 +- 162
5403 +- 201
319 +- 6
141 +- 10
191 +- 2
pPLAM5 ([Delta]C1400)
8679 +- 522
322 +- 4
4026 +- 276
4800 +- 435
394 +- 22
218 +- 18
355 +- 10
pPL1301m26 (G1505U)
7461 +- 428
159 +- 21
2601 +- 143
3647 +- 155
180 +- 10
108 +- 5
147 +- 11
pNOG529U
6487 +- 645
326 +- 11
4161 +- 119
5429 +- 270
478 +- 31
278 +- 96
364 +- 15
pNOU1199C/C1200U
7469 +- 437
574 +- 18
3712 +- 154
4641 +- 475
585 +- 40
365 +- 16
628 +- 27
pNOU1199G/C1200G
9319 +- 568
591 +- 26
4196 +- 159
5169 +- 411
557 +- 16
365 +- 15
629 +- 22
pNOC1192U (wt)
b
8985 +- 589
120 +- 6
1709 +- 350
3486 +- 168
95 +- 5
68 +- 8
118 +- 4
pKKC1192U (wt)
b
17 241 +- 2738
161 +- 21
2579 +- 514
13 139 +- 1351
142 +- 3
107 +- 10
161 +- 7
pKKA792U
b
19 388 +- 1180
214 +- 19
3615 +- 707
12 575 +- 1524
132 +- 32
116 +- 17
178 +- 18
pKKA792G
b
14 176 +- 2237
142 +- 7
2138 +- 474
n.d.
105 +- 4
90 +- 13
131 +- 9
pKKG1530A/A1531G
b
n.d.
167 +- 5
2994 +- 79
13 672 +- 453
148 +- 10
130 +- 14
168 +- 35
a
Numbers represent Miller units of [beta]-galactosidase activity, +- one standard error. Each assay value represents the mean
value for 3-5 independent measurements.
b
Contains, in addition, the U1192 mutation.
n.d., not determined.
Interaction of G1505 with other regions of 16S rRNA
Influence of the
ksgA
-dependent dimethylation of A1518 and A1519 on A and P site decoding
The antibiotic kasugamycin is believed to inhibit protein synthesis by interfering with the ribosomal P site (
34
). However, an effect of kasugamycin on A site decoding has also been reported (
35
). Resistance to the antibiotic can arise through mutations in the KsgA
methylase that modifies A1518 and A1519 near the 3'-end of 16S rRNA. Methylase-deficient
ksgA
-
strains are defective in subunit-subunit and IF3 interactions and display elevated levels of stop codon
readthrough and frameshifting (
35
). The influence of unmodified A1518/A1519 on decoding at the P site was tested
by transforming methylase-deficient
ksgA
-
strains with a range of
lacZ
initiation codon constructs. The data presented in Table
4
show that
ksgA
-
strains also display elevated levels of initiation from non-AUG codons. These data suggest that in addition to changes in the primary
structure, alterations in the pattern of post-transcriptional modification of 16S rRNA can affect decoding fidelity at
both the A and P sites.
.
Effects of ksgA-dependent rRNA modification on initiation from non-AUG codons
LacZ plasmids
Strain/mutation
MC41, wild type
MC178, ksgA19
pSG25-AUG
25 858 +- 597
27 989 +- 829
pSG413-CUG
508 +- 62
868 +- 36
pSG415-AUC
461 +- 71
870 +- 154
pSG416-AUA
501 +- 42
674 +- 93
pSG417-AUU
535 +- 73
835 +- 140
Numbers represent Miller units of [beta]-galactosidase activity (56), and are the mean values of 3-6
independent measurements, +- one standard error.
DISCUSSION
Chemical protection and crosslinking experiments have defined specific bases in
16S rRNA that are involved in tRNA binding to the ribosomal A, P and E sites
and have helped define the orientation of tRNAs on the ribosome (
6
). The data presented here show that mutations at several of these positions,
such as G529, which has been linked to A site function, and C1400, which has
been linked to P site function, affected decoding at both the A and P sites.
This suggests that mutations in the 530 loop, the 1400-1500 region and mutations in helix 34 all lead to a distortion of the
site of codon-anticodon interaction that results in a loss of discrimination between
cognate and near-cognate tRNAs at the adjacent A and P sites. The effect of rRNA mutations
on tRNA selection might be similar to that provoked by the error-inducing aminoglycoside antibiotics, which are thought to enhance non-specific tRNA-ribosome interactions at the expense of accurate codon-anticodon interactions (
36
). Weakening of specific mRNA-tRNA contacts would also be consistent with the effects of the rRNA
mutations on tRNA slippage that lead to reading frame errors (Table
2
and Fig.
2
). In this context, it is significant that the antibiotics streptomycin and
neomycin, as well as error-enhancing mutations in ribosomal proteins S4 and S5 and error-restrictive mutations in ribosomal protein S12, have all been shown
to affect tRNA-ribosome interactions at both the A and P sites (
37
). However, an important functional difference between these ribosomal mutants
is that while the antibiotics and ribosomal protein mutations had differential
effects on tRNA-ribosome interactions at the ribosomal A and P sites, the rRNA mutations
described here had the same error-enhancing effect on decoding at both sites.
The 1400-1500 region of 16S rRNA
Both A and P site-bound tRNAs reduce or enhance the reactivities of several nucleotides in
the 1400-1500 region towards chemical probes (
5
) and aminoglycoside antibiotics that perturb decoding protect A1408, G1491 and
G1494 (
34
). Furthermore, it has been shown that positions +4 and +7 on the mRNA can be
crosslinked to bases C1402 and C1395 respectively (
38
). Consequently, the effects of the 1400-1500 region mutations on decoding are likely to derive from direct
disruption of tRNA-mRNA-ribosome interactions. Although the nucleotides surrounding C1400
were originally depicted as being single stranded, recent phylogenetic and
mutational analyses have suggested that nucleotides adjacent to C1400 are base
paired with nucleotides adjacent to A1500 (
39
; Fig.
1
). Thus, C1407 is proposed to be paired with G1494 and genetic evidence has been
obtained for the existence of G1401-C1501, C1404-G1497 and G1405-C1496 base pairs (
40
,
41
). In the C1407U mutation analyzed here, for instance, a U-G pair has replaced the native C-G pair. Analyses of ribosomes carrying disruptions in tertiary
base pairs or deletions in the 1400-1500 region, by Ofengand and co-workers, indicated that initiation, as well as binding of tRNA to A
and P sites, was affected by the mutations (
40
-
42
). This is consistent with our results, which show that disruption of the
decoding center by the C1395U, [Delta]C1400, C1407U and G1505U mutations influences maintenance of the reading
frame and affects tRNA-mRNA-ribosome interactions at the adjacent A and P sites.
Helix 34
The isolation of an apparently UGA-specific suppressor mutation at position C1054 in
E.coli
16S rRNA led to the elaboration of a model for UGA termination (
43
). This model proposed that recognition of the UGA termination codon occurred
through base pairing between the UGA triplet and either of the tandem UCA
triplets at bases 1199-1204 in helix 34. However, subsequent analyses demonstrated that
mutations at or adjacent to positions C1054/C1200 caused readthrough of all
three stop codons, as well as enhancing frameshifting levels (
11
). In this study we have demonstrated that the effects of mutations in helix 34
are not limited to the elongation phase of translation, but also affect the
fidelity of decoding in the P site during initiation. Another rRNA-mRNA base pairing model for termination was proposed by Tate
et al.
(
44
), based on crosslinking of the UAA termination triplet to position A1408.
However, the data presented here show that the C1407U mutation, which alters
the proposed recognition sequence for the termination triplets, not only
affected readthrough of all three stop codons and frameshifting, but also affected the fidelity of decoding at initiation. Moreover, mutations at C1395, C1400 and G1505 provoked the same pattern of A site and P site
decoding errors as was observed with the C1407U mutation. While these results
underline the importance of helix 34 and the C1400 region in reading frame
maintenance and tRNA-mRNA interactions in both the A and P sites, they do not support the
specific involvement of either of these regions in termination. Consequently,
the effects of mutations in both of these regions of 16S rRNA most likely
derive from disruption of tRNA-mRNA-ribosome interactions in a general way, rather than from a
specific influence on the termination process. Direct evidence linking helix 34
with mRNA-ribosome interactions came from site-directed crosslinking experiments, which showed that while
positions +4 and +7 of the mRNA were crosslinked to C1395 and C1402
respectively, position +6 was crosslinked to U1052 in helix 34 (
38
,
45
). In addition tetracycline, an antibiotic that inhibits A site tRNA binding,
protects A892 in the central domain and U1052 and C1054 in helix 34 from
chemical modification (
46
). Thus, while there are no tRNA-dependent crosslinks or chemical protections in helix 34, the mRNA
crosslinks and antibiotic footprints, together with the genetic data presented
here, indicate the involvement of helix 34 in decoding functions at initiation
and elongation.
The 530 loop
Genetic evidence linking the 530 loop with decoding function came from isolation
of a nonsense suppressor mutation at position G517 in yeast mitochondria (
47
) and mutations conferring resistance to the error-inducing antibiotic streptomycin in chloroplasts and mycobacteria (
48
,
49
). The involvement of the 530 loop in decoding and antibiotic resistance in both
E.coli
and yeast cytoplasmic ribosomes was subsequently established by site- directed mutagenesis of rRNA operons (
10
,
16
,
50
,
51
). Analysis of a lethal G530A mutation by Powers and Noller (
52
) suggested that these mutant ribosomes might be specifically defective in EF-Tu-ribosome interactions. In addition, it was found that antibiotics
and ribosomal protein mutations that promoted miscoding differentially affected
tRNA-dependent protection of G530, but not protection of A1492/A1493 at the A
site (
53
). These findings led to the advancement of a model which proposes that the
conformation of the 530 loop responds differentially to cognate versus non-cognate ternary complex binding during initial recognition and proofreading of tRNAs (
52
,
53
). According to this model, the 530 loop influences tRNA binding via its effects on ribosome-EF-Tu interaction. However, our results indicate that the 530 loop may
play a much more direct role in tRNA-ribosome interaction and at multiple stages of translation. The G529U
mutation affects reading frame maintenance and decoding fidelity during both
the initiation and elongation phases of translation. Initiator tRNA binding to
the ribosomal P site does not involve EF-Tu and at least some of the ribosomal frameshifting events that are influenced by mutations in the 530 loop involve slippage
of cognate tRNAs in the P site (rather than mistranslocation of non-cognate tRNAs or out of phase tRNA binding to the A site). These
considerations, together with the functional similarity of the G529U mutation
to changes in helix 34 and the C1400 region, suggest that all three regions of
rRNA comprise a decoding domain and that alterations in any of the three regions have similar effects on tRNA-ribosome interactions. This interpretation is consistent with the mRNA crosslinking data, which show that
the 530 loop, helix 34 and the C1400 region all form crosslinks to mRNA between
positions +4 and +11 (
38
,
45
,
54
). Also consistent with this arrangement of the decoding domain are the recent
data of Heilek and Noller (
55
), who have determined the rRNA neighborhood of ribosomal protein S5 using a
Fe(II)-EDTA cleavage reagent tethered to amino acid 21. Using this reagent,
cleavages were observed in the 420 and 530 loops, the central pseudoknot, the
920 and 1400 regions and in helix 34, indicating the close physical proximity
of all of these regions of 16S rRNA.
ACKNOWLEDGEMENTS
We are indebted to Edward Meenan, Drs John Atkins and Ray Gesteland for carrying
out protein sequencing analyses, Stephen Lodmell and Steven Gregory for
constructing some of the pSG plasmids and Barbara Bachmann, Melvin Santer and
Matthew Firpo for providing plasmids and bacterial strains. We thank Dr George
Q.Pennabble for his erudite insights, David Jemiolo for sharing unpublished
results and our colleagues in the Dahlberg and Zimmermann laboratories for
their comments on this manuscript. This work was supported by grants GM19756 to
A.E.D and GM22807 to R.A.Z from the National Institutes of Health.
3 Prince,J.B., Taylor,B.H., Thurlow,D.L., Ofengand,J. and Zimmermann,R.A. (1982) Proc. Natl. Acad. Sci. USA, 79, 5450-5454.MEDLINE Abstract
4 Purohit,P. and Stern,S. (1994) Nature, 377, 659-662.
5 Moazed,D. and Noller,H.F. (1990) J. Mol. Biol., 211, 135-145.MEDLINE Abstract
6 Brimacombe,R. (1995) Eur. J. Biochem., 182, 501-506
7 Zimmermann,R.A. (1995) In Zimmermann,R.A. and Dahlberg,A.E. (eds), Ribosomal RNA, Structure, Evolution, Processing and Function in Protein Biosynthesis. CRC Press, Boca Raton, FL, pp. 277-309.
8 O'Connor,M., Brunelli,C.A., Firpo,M.A., Gregory,S.T., Lieberman,K.R., Lodmell,J.S., Moine,H., Van Ryk,D.I. and Dahlberg,A.E. (1995) Biochem. Cell Biol., 73, 859-868.
9 Ofengand,J.,Bakin,A. and Nurse,K. (1993) In Nierhaus,K.H., Franchesi,F., Subramanian,A.R., Erdmann,V.A. and Wittmann-Liebold,B. (eds), The Translational Apparatus. Structure, Function, Regulation, Evolution. Plenum Press, New York, NY, pp. 489-500.
35 van Buul,C.P.J.J., Visser,W. and van Knippenberg,P.H. (1984) FEBS Lett., 177, 119-124.
36 Cundliffe,E. (1981) In Gale,E.F., Cundliffe,E., Reynolds,E., Reynolds,P.E., Richmond,M.H. and Waring,M.J. (eds), The Molecular Basis of Antibiotic Action. Wiley, New York, NY, pp. 402-547.
37 Karimi,R. and Ehrenberg,M. (1996) EMBO J., 15, 1149-1154.MEDLINE Abstract
