![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
516 and A535 in Escherichia coli 16S rRNA1 ,2
Department of Biological Sciences, Wayne State University, Detroit, MI 48202
5To whom correspondence should be addressed. E-mail: philc{at}wayne.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
|---|
) in Escherichia coli 16S rRNA which is located at
position 516 of the conserved 530 loop. The nucleotide opposite 516
in the hairpin, A535, was also mutated. The data show that a pyrimidine
( or C) is required at position 516, while substitutions at position
535 reduce ribosome function by < 50%. A requirement for base
pair formation between 516 and A535 was not indicated.
KEY WORDS: • ribosomal RNA • protein synthesis • 530 loop • pseudouridine • mutational analysis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
|---|
). Genetic and functional analyses of
rRNA mutations in Escherichia coli and most other organisms
have been complicated by the presence of multiple rRNA genes and by the
occurrence of dominant lethal rRNA mutations. Because there are seven
rRNA operons in E. coli, the phenotypic expression of rRNA
mutations may be affected by the relative amounts of mutant and
wild-type ribosomes in the cell. Thus, detection of mutant
phenotypes can be hindered by the presence of wild-type ribosomes.
A variety of approaches have been designed to circumvent these
problems. One common approach uses cloned copies of a wild-type
rRNA operon (2
, 3
). Several groups have used this system to
detect phenotypic differences caused by high level expression of mutant
ribosomes. Recently, a strain of E. coli was constructed in
which the only supply of ribosomal RNA was plasmid encoded
(4
). This system has been used to study transcriptional
regulation of rRNA synthesis (5
) as well as ribosomal RNA
function (5
–10
). Hui et al. (11
) showed that
mRNA could be directed to a specific subset of plasmid-encoded
ribosomes by altering the message binding site
(MBS)6
of the ribosome while at the same time altering the ribosome binding
site (RBS) of an mRNA. Although each of the above methods has contributed significantly to our understanding of rRNA function, progress in this field has been hampered both by the complexity of translation and by difficulty in applying standard genetic selection techniques to these systems. Here, we describe the development of a stable genetic system that allows direct selection and analysis of rRNA primary mutations that would normally be lethal and isolation of second-site complementation mutants.
This system was used to investigate the functional and structural role
of the single pseudouridine () located at position 516 in E.
coli 16S rRNA. This modified nucleoside is part of a highly
conserved hairpin, the 530 loop, found in all small ribosomal subunit
RNA (12
) (Fig. 1
). This stem loop contains two modified nucleosides, 516 and
m7G527, and two pseudoknot structures between
residues 524–526 and 505–507 and residues 521–522 and 527–528 which
contribute to the complexity of its structure (13
–17
).
Nucleotides in this hairpin have been implicated in tRNA binding
(18
–20
), translational fidelity (21
–24
),
streptomycin resistance (15
, 25
–27
) and protein binding
(28
–32
).
|
). Phylogenetic studies suggest that the presence of
pseudouridine at this position is specific to bacteria
(33
, 34
). Despite its abundance in RNA, little is known
about the effect of pseudouridine on RNA structure or its role in
ribosome function. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
|---|
All reagents and chemicals were as in (43
).
PCR-directed mutagenesis was performed essentially by the method of
Higuchi (35
) and the primers used are listed in
;A1>. Plasmids used in this study are listed
in ;A2>.
Bacterial strains and media.
All plasmids were maintained and expressed in E.
coli DH5 (supE44, hsdR17, recA1, endA1, gyrA96,
thi-1 and relA1) (36
). To induce
synthesis of plasmid-derived rRNA from the lacUV5
promoter, IPTG was added to a final concentration of 1 mM.
Chloramphenicol acetyltransferase activity was determined essentially
as described by Nielsen et al. (37
). Cultures for CAT
assays were grown in LB-Ap100. MIC were determined by standard
methods in microtiter plates as described in (44
).
Primer extension.
To determine the ratio of plasmid to chromosome-derived rRNA,
pRNA104 containing cells growing in LB-Ap100 were harvested at the time
intervals indicated and total RNA was extracted using the Qiagen RNeasy
kit (Chatsworth, CA). The 30S, 70S and crude ribosomes were
isolated from 200 mL of induced, plasmid containing cells by the method
of Powers and Noller (15
). The purified RNA was analyzed
by primer extension according to Sigmund et al. (38
).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
|---|
An overview of the steps used to construct the system is shown in
Figure 2
. The key features of the initial construct, pRNA9, are: 1)
it contains a copy of the rrnB operon from pKK3535
(2
) under transcriptional regulation of the
lacUV5 promoter; this well-characterized promoter is not
subject to catabolic repression and is easily and reproducibly
inducible with isopropyl-ß-D-thiogalactoside
(IPTG). 2) To minimize transcription in the absence of
inducer, PCR was used to amplify and subclone the lac
repressor variant, lacIq (39
)
from pSPORT1 (Life Technologies, Rockville, MD). (3) The
chloramphenicol acetyltransferase gene (cam) is present and
transcribed constitutively from a mutant tryptophan promoter,
trpc (40
, 41
). 4)
The ß-lactamase gene is also present to allow maintenance of plasmids
in the host strain. To allow genetic selection, the CAT structural gene
from pJLS1021 (42
) was amplified and placed downstream of
a constitutive trpc promoter using PCR.
Expression of the CAT gene in E. coli renders the cell
resistant to chloramphenicol and the minimal inhibitory concentration
(MIC) of chloramphenicol increases proportionally with the amount of
CAT protein produced (43
, 44
).
|
To isolate MBS-RBS combinations that are nonlethal and efficiently
translated only by plasmid-derived ribosomes, we used a random
mutagenesis and selection scheme similar to that described by Lee et
al. (43
). The plasmid-encoded 16S MBS and CAT RBS were
randomly mutated using PCR so that the wild-type nucleotide at each
position was excluded (Fig. 3
). The resulting 2.5 x 106 doubly mutated
transformants were induced for 3.5 h in SOC medium
containing 1 mM IPTG and plated on Luria broth medium
containing 100 µg/mL ampicillin, 350 µg/mL chloramphenicol and 1 mM
IPTG. To confirm the presence of all three alternative nucleotides at
each mutated position, plasmid DNA from 
2.0 x 105 transformants was sequenced (Fig. 3)
. The
data show that all of the nonexcluded nucleotides were equally
represented in the random pool. Of the 2.5 x 106 transformants plated, 536 survived the
chloramphenicol selection. The efficiency of the selected MBS-RBS
combinations was determined by measuring the MIC of chloramphenicol for
each survivor in the presence and absence of inducer ();A3> (43
, 44
). Nine of the isolates (1.7%)
showed MIC in the presence of inducer, which were lower than the 350
µg/mL concentration at which they were selected. These were slow
growing mutants that appeared after 48 h during the initial
isolation. The MIC, however, were scored after only 24 h. The MIC
for 451 of the isolates (84.1%) were between 400 and 600 µg/mL, and
the remaining 76 clones (14.2%) were > 600 µg/mL. The
difference in chloramphenicol resistance between induced and uninduced
cells (
MIC) is the amount of CAT translation by plasmid-derived
ribosomes only. A specific interaction between plasmid-derived
ribosomes and CAT mRNA was indicated in 79 (14.7%) of the clones,
which showed four- to eightfold increases in CAT resistance upon
addition of IPTG . Based on these analyses, 11 clones were
retained for additional study. The MBS and RBS in plasmids from these
clones were sequenced and CAT assays and growth curves were performed
( and Fig. 4
).
|
|
G°37). Purines were preferred in all of the
MBS positions but the RBS did not show this sort of selectivity. This
can be explained partially by the observation that the selected RBS can
base pair with sequences adjacent to the mutated region of 16S rRNA as
described by Lee et al. (43
).
Growth curves were performed for all of the selected mutants and
compared with strains containing control constructs (Fig. 4)
. Only one
mutant (IX24) is shown in Figure 4
, but all strains containing the
selected MBS/RBS sequences showed the same pattern of growth as this
mutant. Because of its induction profile, strain IX24 (containing
plasmid pRNA100) was chosen for additional experimentation.
To eliminate the possibility that mutations outside the MBS and RBS had
been inadvertently selected, the DraIII and XbaI
fragment containing the MBS and the KpnI and XhoI
fragment containing the RBS sequence from pRNA100 (Fig. 5
) were transferred to pRNA9.
|
The rate of ribosome induction and the ratio of plasmid to
chromosome-derived rRNA at each stage of growth were determined.
For this, a pRNA100 derivative, pRNA104, which contains a C1192U
mutation in 16S rRNA was constructed (45
, 46
) so that
plasmid-derived rRNA could be differentiated from wild-type
rRNA by primer extension. The C1192U mutation does not affect ribosome
function in other expression systems (45
, 47
). To show that
the same is true in our system, CAT activity was measured after 3 h induction with 1 mM IPTG in DH5 cells expressing pRNA100 or pRNA104
and the two were compared. In these experiments, no significant
difference between cells expressing pRNA104 (99.2 ± 2.8%) or
pRNA100 (100%) was observed.
Next, to determine the percentage of plasmid-derived ribosomes in
cells containing the plasmid, total RNA was isolated from DH5 cells
carrying pRNA104 before and after induction with IPTG and subjected to
primer extension analysis (44
, 45
, 47
). Maximum induction of
plasmid-derived ribosomes occurred 3 h after induction at
which point they constituted 40% of the total ribosome pool
(Fig. 6
). CAT activities in these cells paralleled induction of
plasmid-derived ribosomes and began to decrease 4 h after
induction, presumably due to protein degradation during stationary
phase. In uninduced cells, 3% of the total ribosome pool contains
plasmid-derived ribosomes because of basal level transcription from
the lacUV5 promoter.
|
Chloramphenicol resistance in uninduced cells containing pRNA100 is 75
µg/mL (MIC = 100 µg/mL; );A5>. By
measuring CAT resistance in a derivative of pRNA100 containing a
wild-type 16S rRNA gene, it was determined that approximately
one-half of this background activity was due to CAT translation by
wild-type ribosomes (; pRNA100 + wt MBS). The remaining
activity in uninduced cells is presumably due to leakiness of the
lacUV5 promoter (Fig. 6)
. The nucleotide sequence located
between the RBS and the start codon in mRNA affects translational
efficiency (39
, 48
, 49
). In pRNA100, three of the
nucleotides found in this region of the CAT mRNA are complementary with
the 3' terminus of wild-type E. coli 16S RNA (, pRNA100 + wt MBS). To eliminate the possibility that this
was contributing to CAT translation in the absence of
plasmid-encoded ribosomes, four nucleotides in the CAT gene
(underlined in ) were randomly mutagenized and screened to
identify mutants with reduced translation by host ribosomes. A total of
2000 clones were screened in the absence of plasmid-encoded
ribosomes using pCAM9 and six poorly translated CAT sequences were
isolated (Fig. 5)
. Next, the BamHI fragment of pRNA100
containing lacIq and the rrnB
operon was added, and MIC, CAT assays and growth curves were performed
on cells expressing these constructs (data not shown). Based on these
data, pRNA122 was chosen because it produced a slightly better
induction profile than the others . Translation of the
pRNA122 CAT message by wild-type ribosomes (; pRNA122 +
wt MBS) produces cells that are sensitive to chloramphenicol
concentrations < 10 µg/mL. In the presence of specialized
ribosomes (; pRNA122), the background chloramphenicol MIC is
between 40 and 50 µg/mL and the MIC for induced cells is between 550
and 600 µg/mL, producing an 13-fold increase in CAT expression
upon induction in pRNA122. Induction of the rrnB operon in
pRNA100 produces only an eightfold increase.
Use of the system.
To test the system, we examined the effects of nucleotide substitutions
at the sole pseudouridine in E. coli 16S rRNA, located at
position 516. Because and U form equally stable base pairs with
adenosine (50
), we also constructed mutations at
A535 to see whether the potential for base pair formation between these
two loci affected ribosome function. The mutations were constructed
initially in a pUC19 (51
) derivative containing the 16S
RNA gene, p16ST, as shown in Figure 7
and then transferred to pRNA122 for analysis. This two-step process
was used because the SacII restriction site located between
the two mutated positions is unique in pRNA16ST and is not unique in
pRNA122. The effect of the mutations in pRNA122 on protein synthesis in
vivo was determined by measuring the MIC and CAT activity of the mutant
cells (Fig. 8
). At position 516, ribosomes containing the single transition mutation,
516C, produced 60% of the amount of functional CAT protein
produced by wild-type ribosomes. The transversion mutations,
516A or 516G, however, reduced ribosome function by > 90%.
All of the single mutations at position 535 retained > 50% of
the function of wild-type ribosomes. To examine the possibility
that the potential for base pairing between positions 516 and 535 is
necessary for ribosome function, all possible mutations between these
loci were also constructed and analyzed (Fig. 8)
. These data show that
all of the double mutants were inactive (10% or less of the wild-type)
regardless of the potential to base pair. To examine the reasons for
loss of function in the 516 mutants, ribosomes from cells expressing
single mutations at position 516 were fractionated by sucrose density
gradient centrifugation and the 30S and 70S peaks were analyzed by
primer extension to determine the percentage of plasmid-derived 30S
subunits present. The data in show a
strong correlation between ribosome function and the presence of
plasmid-derived ribosomes in the 70S ribosomal fraction, indicating
that mutations at positions 516 affect the ability of the mutant 30S
subunits to form 70S ribosomes.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
|---|
Our initial studies on the sequence constraints of the
Shine-Dalgarno interaction (35
) showed that through
random mutagenesis of the MBS and RBS alternative functional
combinations could be selected. In this study we described the use of
this approach to identify and refine alternative MBS-RBS
combinations that allow efficient and specific translation of
nonchromosomal messages. In this system, plasmid-derived ribosomes
comprise 45% of the total ribosome population because the
rrnB operon is transcribed from the lacUV5
promoter. To reduce background expression in our original construct,
pRNA100, a new sequence was introduced upstream of the CAT gene and
resulted in very low CAT translation by host ribosomes. In the absence
of inducer, pRNA122 renders cells resistant to 40 µg of
chloramphenicol per milliliter. Because approximately one-half of
this background is due to translation by plasmid-derived ribosomes
and induction with IPTG makes cells resistant to 550 µg/mL,
this system allows genetic selection of mutants with a wide range of
function . Thus, the stability, low background and high
level of chloramphenicol resistance upon induction in cells
carrying pRNA122 provide an ideal genetic system for the study of
ribosomal RNA structure and function. A key aspect of this system
is the ability to select and analyze stable ribosomal RNA mutations
in vivo that are only partially functional or that would normally
be lethal.
Investigation of the function of 516 in protein synthesis.
Several studies suggest that the 530 stem loop has a higher order
structure and interacts with EF-Tu·GTP·aminoacyl tRNA ternary
complex and ribosomal proteins S4 and S12, which have been shown to be
involved in translational fidelity. Two pseudoknots and two modified
nucleosides are present in this region and have been implicated in
establishing the higher order structure and interacting with
translational factors. The only pseudouridine in 16S RNA is located at
position 516 in 16S rRNA and seems to be specific to bacteria. Because
is unique, we constructed mutations at this site to determine which
types of nucleotides could be substituted while maintaining function.
Although no indication of pairing with A535 is found in the
phylogenetic data, we also mutated this site to see whether any
interaction were possible between these two loci. Figure 8
shows a
summary of mutational analyses at positions 516 and 535. These data
indicate that the potential to base pair between positions 516 and 535
does not affect function but that base identity ( or C) at position
516 is critical. The most active mutant was the single A535U mutant,
which is also the most common phylogenetic variant (16
).
Deletion of the rsuA gene, the pseudouridine synthase that
converts U516 to pseudouridine in 16S ribosomal RNA of E.
coli, does not affect cell growth at varying temperatures
(52
). This suggests that either pseudouridine or uridine
at position 516 is sufficient to permit protein synthesis under
normal growth conditions. Because our data show that transition
mutations are less inhibitory than transversion mutations, it seems
likely that 516 is not directly involved in ribosome function but,
instead, may stabilize the conformation of the 530 loop. It is possible
that when examined under other environmental or nutritional conditions,
a more pronounced effect on cell viability may be observed in the
rsuA mutant (53
).
The subunit association data showed that mutations at position 516
affect subunit association . It is unclear, however,
whether this is due to direct involvement of this residue with some
portion of the 50S subunit or due indirectly to a change in structure
or interruption of the initiation pathway leading to formation of the
70S ribosome. It is interesting to note that all of the observed loss
of function in the 516C mutant can be explained by inability of the
mutant subunits to form a 70S complex. Substitution of a purine,
however, produces ribosomes that are less functional than predicted
from the subunit association data. These results show that all
mutations at 516 inhibit subunit association and suggest that the
purine substitutions either disrupt an additional aspect of protein
synthesis or perturb the structure at the subunit interface when
present in 70S ribosomes (20
).
|
APPENDIX |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
|---|
|
|
|
|
|
|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Supported by National Institutes of Health
Grants GM55745 and GM52896.
3 Present address: Department of Genetics,
Stanford University School of Medicine, Stanford, CA 94305.
4 Present address: Henry Ford Health Systems,
Infectious Disease Research, E&R 7045, 2799 W. Grand River Blvd.,
Detroit, MI 48202.
6 Abbreviations used: IPTG,
isopropyl-ß-D-thiogalactoside; MBS, message binding site;
MIC, minimal inhibitory concentration; RBS, ribosome binding site.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
|---|
1. Garrett, R. A. Douthwaite, S. R. Liljas, A. Matheson, A. T. Moore, P. B. Noller, H. F. eds. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions 2000 ASM Press Washington, DC. .
2. Brosius, J., Ullrich, A., Raker, M. A., Gray, A., Dull, T. J., Gutell, R. R. & Noller, H. F. (1981) Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid 6:112-118.
3. Sigmund, C. D. & Morgan, E. A. (1982) Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 79:5602-5606.
4. Asai, T., Condon, C., Voulgaris, J., Zaporojets, D., Shen, B., Al-Omar, M., Squires, C. & Squires, C. L. (1999) Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons. J. Bacteriol. 181:3803-3809.
5. Voulgaris, J., French, S., Gourse, R. L., Squires, C. & Squires, C. L. (1999) Increased rrn gene dosage causes intermittent transcription of rRNA in Escherichia coli. J. Bacteriol. 181:4170-4175.
6. Koosha, H., Cameron, D., Andrews, K., Dahlberg, A. E. & March, P. E. (2000) Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene. RNA 6:1166-1173.[Abstract]
7. Sergiev, P. V., Bogdanov, A. A., Dahlberg, A. E. & Dontsova, O. (2000) Mutations at position A960 of E coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA. J. Mol. Biol. 299:379-389.[Medline]
8. O’Connor, M. & Dahlberg, A. E. (2001) Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA. Nucl. Acids Res. 29:1420-1425.
9. O’Connor, M., Lee, W. M., Mankad, A., Squires, C. L. & Dahlberg, A. E. (2001) Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable. Nucl. Acids Res. 29:710-715.
10. Vila-Sanjurjo, A. & Dahlberg, A. E. (2001) Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction. J. Mol. Biol. 308:457-463.[Medline]
11. Hui, A., Jhurani, P. & de Boer, H. A. (1987) Directing ribosomes to a single mRNA species: a method to study ribosomal RNA mutations and their effects on translation of a single messenger in Escherichia coli. Methods Enzymol. 153:432-452.[Medline]
12. Gutell, R. R., Larsen, N. & Woese, C. R. (1994) Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol. Rev. 58:10-26.
13. Zueva, V. S., Mankin, A. S., Bogdanov, A. A. & Baratova, L. A. (1985) Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA. Eur. J. Biochem. 146:679-687.[Medline]
14. Woese, C. R. & Gutell, R. R. (1989) Evidence for several higher order structural elements in ribosomal RNA. Proc. Natl. Acad. Sci. U.S.A. 86:3119-3122.
15. Powers, T. & Noller, H. F. (1991) A functional pseudoknot in 16S ribosomal RNA. EMBO J 10:2203-2214.[Medline]
16. Gutell, R. R. (1993) The simplicity behind the evolution of complex structure in ribosomal RNA. Nierhaus, K. H. Franceschi, F. Subramanian, A. R. eds. The Translational Apparatus: Structure, Function, Regulation, Evolution 1993:477-488 Plenum Press New York, NY. .
17. Bakin, A., Kowalak, J. A., McCloskey, J. A. & Ofengand, J. (1994) The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516. Nucleic Acids Res 22:3681-3684.
18. Moazed, D. & Noller, H. F. (1990) Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA. J. Mol. Biol. 211:135-145.[Medline]
19. Ogle, J. M., Brodersen, D. E., Clemons, W. M., Jr, Tarry, M. J., Carter, A. P. & Ramakrishnan, V. (2001) Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292:897-902.
20. Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Earnest, T. N., Cate, J. H. & Noller, H. F. (2001) Crystal structure of the ribosome at 5.5 A resolution. Science 292:883-896.
21. Thompson, R. C. (1988) EFTu provides an internal kinetic standard for translational accuracy. Trends Biochem. Sci. 13:91-93.[Medline]
22. O’Connor, M., Goringer, H. U. & Dahlberg, A. E. (1992) A ribosomal ambiguity mutation in the 530 loop of E coli. 16S rRNA. Nucleic Acids Res 20:4221-4227.
23. Powers, T. & Noller, H. F. (1994) Selective perturbation of G530 of 16 S rRNA by translational miscoding agents and a streptomycin-dependence mutation in protein S12. J. Mol. Biol. 235:156-172.[Medline]
24. Santer, U. V., Cekleniak, J., Kansil, S., Santer, M., O’Connor, M. & Dahlberg, A. E. (1995) A mutation at the universally conserved position 529 in Escherichia coli 16S rRNA creates a functional but highly error prone ribosome. RNA 1:89-94.[Abstract]
25. Gauthier, A., Turmel, M. & Lemieux, C. (1988) Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: evidence for a novel site of streptomycin resistance in the small subunit rRNA. Mol. Gen. Genet. 214:192-197.[Medline]
26. Melancon, P., Lemieux, C. & Brakier-Gingras, L. (1988) A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin. Nucleic Acids Res 16:9631-9639.
27. Santer, M., Santer, U., Nurse, K., Bakin, A., Cunningham, P., Zain, M., O’Connell, D. & Ofengand, J. (1993) Functional effects of a G to U base change at position 530 in a highly conserved loop of Escherichia coli 16S RNA. Biochemistry 32:5539-5547.[Medline]
28. Ozaki, M., Mizushima, S. & Nomura, M. (1969) Identification and functional characterization of the protein controlled by the streptomycin-resistant locus in E coli. Nature 222:333-339.[Medline]
29. Bock, A., Petzet, A. & Piepersberg, W. (1979) Ribosomal ambiguity (ram) mutations facilitate diyhydrostreptomycin binding to ribosomes. FEBS Lett 104:317-321.[Medline]
30. Stern, S., Wilson, R. C. & Noller, H. F. (1986) Localization of the binding site for protein S4 on 16 S ribosomal RNA by chemical and enzymatic probing and primer extension. J. Mol. Biol. 192:101-110.[Medline]
31. Capel, M. S., Engelman, D. M., Freeborn, B. R., Kjeldgaard, M., Langer, J. A., Ramakrishnan, V., Schindler, D. G., Schneider, , D. K., Schoenborn, B. P., Sillers, I. Y., Yabuki, S. & Moore, P. B. (1987) A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli. Science 238:1403-1406.
32. Stern, S., Powers, T., Changchien, L. M. & Noller, H. F. (1989) RNA-protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA. Science 244:783-790.
33. Wrzesinski, J., Bakin, A., Nurse, K., Lane, B. G. & Ofengand, J. (1995) Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli. Biochemistry 34:8904-8913.[Medline]
34. Bakin, A. & Ofengand, J. (1995) Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution. Nucleic Acids Res 23:3290-3294.
35. Higuchi, R. (1989) Erlich, H. A. eds. Using PCR to engineer DNA, in PCR Technology 1989:61-70 Stockton Press New York, NY .
36. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.[Medline]
37. Nielsen, D. A., Chang, T. C. & Shapiro, D. J. (1989) A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells. Anal. Biochem. 179:19-23.[Medline]
38. Sigmund, C. D., Ettayebi, M., Borden, A. & Morgan, E. A. (1988) Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli. Methods Enzymol 164:673-690.[Medline]
39. Calos, M. P. (1978) DNA sequence for a low-level promoter of the lac repressor gene and an ’up’ promoter mutation. Nature 274:762-765.[Medline]
40. De Boer, H. A., Comstock, L. J. & Vasser, M. (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. U.S.A. 80:21-25.
41. Hui, A. & De Boer, H. A. (1987) Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 84:4762-4766.
42. Schottel, J. L., Sninsky, J. J. & Cohen, S. N. (1984) Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system. Gene 28:177-193.[Medline]
43. Lee, K., Holland-Staley, C. A. & Cunningham, P. R. (1996) Genetic analysis of the Shine-Dalgarno interaction: selection of alternative functional mRNA-rRNA combinations. RNA 2:1270-1285.[Abstract]
44. Lee, K., Varma, S., SantaLucia, J., Jr & Cunningham, P. R. (1997) In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA. J. Mol. Biol. 269:732-743.[Medline]
45. Sigmund, C. D., Ettayebi, M. & Morgan, E. A. (1984) Antibiotic resistance mutations in 16S and 23S ribosomal RNA genes of Escherichia coli. Nucleic Acids Res. 12:4653-4663.
46. Triman, K., Becker, E., Dammel, C., Katz, J., Mori, H., Douthwaite, S., Yapijakis, C., Yoast, S. & Noller, H. F. (1989) Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli. J. Mol. Biol. 209:645-653.[Medline]
47. Makosky, P. C. & Dahlberg, A. E. (1987) Spectinomycin resistance at site 1192 in 16S ribosomal RNA of E. coli: an analysis of three mutants. Biochimie 69:885-889.[Medline]
48. Stormo, G. D., Schneider, T. D. & Gold, L. M. (1982) Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 10:2971-2996.
49. Chen, H., Bjerknes, M., Kumar, R. & Jay, E. (1994) Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucl. Acids Res. 22:4953-4957.
50. Maden, B. E. (1990) The numerous modified nucleotides in eukaryotic ribosomal RNA. Prog. Nucleic Acid Res. Mol. Biol. 39:241-303.[Medline]
51. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.[Medline]
52. Conrad, J., Niu, L., Rudd, K., Lane, B. G. & Ofengand, J. (1999) 16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases. RNA 5:751-763.[Abstract]
53. Charette, M. & Gray, M. W. (2000) Pseudouridine in RNA: what, where, how, and why. IUBMB Life 49:341-351.[Medline]
54. Shen, Z. H. & Fox, T. D. (1989) Substitution of an invariant nucleotide at the base of the highly conserved ’530-loop’ of 15S rRNA causes suppression of yeast mitochondrial ochre mutations. Nucleic Acids Res 17:4535-4539.
55. Stern, S., Powers, T., Changchien, L. M. & Noller, H. F. (1988) Interaction of ribosomal proteins S5 1988:S6 S11 S12, S18 and S21 with 16 S rRNA. J. Mol. Biol. 201 683–695. .
56. Dontsova, O., Dokudovskaya, S., Kopylov, A., Bogdanov, A., Rinke-Appel, J., Junke, N. & Brimacombe, R. (1992) Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region: a site-directed cross-linking study with mRNA analogues. EMBO J 11:3105-3116.[Medline]
57. Brimacombe, R., Mitchell, P., Osswald, M., Stade, K. & Bochkariov, D. (1993) Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA. FASEB J 7:161-167.[Abstract]
58. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467.
This article has been cited by other articles:
|
|
 |
|
  H.-M. Kim, S.-M. Ryou, W.-S. Song, S.-H. Sim, C.-J. Cha, S. H. Han, N.-C. Ha, J.-H. Kim, J. Bae, P. R. Cunningham, et al. Genetic Analysis of the Invariant Residue G791 in Escherichia coli 16S rRNA Implicates RelA in Ribosome Function J. Bacteriol., April 1, 2009; 191(7): 2042 - 2050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Taliaferro and P. J. Farabaugh Testing constraints on rRNA bases that make nonsequence-specific contacts with the codon*anticodon complex in the ribosomal A site RNA, August 1, 2007; 13(8): 1279 - 1286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Gagnon, A. Mukhopadhyay, and S. V. Steinberg Close Packing of Helices 3 and 12 of 16 S rRNA Is Required for the Normal Ribosome Function J. Biol. Chem., December 22, 2006; 281(51): 39349 - 39357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tamura, K. Lee, C. A. Miller, C. J. Moore, Y. Shirako, M. Kobayashi, and S. N. Cohen RNase E Maintenance of Proper FtsZ/FtsA Ratio Required for Nonfilamentous Growth of Escherichia coli Cells but Not for Colony-Forming Ability. J. Bacteriol., July 1, 2006; 188(14): 5145 - 5152. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
) or P site (
