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At least 757 records · Page 42Linked to original sources
PubMed2002

Elongation factor G participates in ribosome disassembly by interacting with ribosome recycling factor at their tRNA-mimicry domains.

Elongation factor G (EF-G) is a G protein with motor function that drives two target molecules, a tRNA in the translating ribosome and the ribosome recycling factor (RRF) in the post-termination complex. How G protein motor action is transmitted to RRF is unknown. Thermus thermophilus RRF is nonfunctional in Escherichia coli. It became functional upon introducing a plasmid expressing E. coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E. coli EF-G tRNA-mimic domain by the Thermus domain. Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region. These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles. These mutational studies suggest that EF-G motor action is transmitted to RRF by specific surface contacts between the domains that mimic the anticodon arm.

Amino Acid Sequence↗
PubMed1994

A photolabile oligodeoxyribonucleotide probe of the decoding site in the small subunit of the Escherichia coli ribosome: identification of neighboring ribosomal components.

In this work we report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 1397-1405 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA. Nucleotides 1397-1405 lie within a single-stranded sequence that has been linked to the decoding region of Escherichia coli ribosomes. On photolysis in the presence of activated 30S subunits, the photolabile oligodeoxyribonucleotide probe site-specifically incorporates into proteins S1, S7, S18, and S21 (identified by SDS-PAGE, RP-HPLC, and antibody affinity chromatography) and into three separate 16S rRNA regions, specifically, nucleotides A-1396, G-1405-A-1408, and A-1492 and A-1493. These results provide clear evidence that G-1405 in 16S rRNA is within 24 A (the distance between G-1405 and the photogenerated nitrene) of proteins S1, S7, S18, and S21 and each of the other nucleotides mentioned above, consistent with other studies of 30S internal structure. Although the probe binds to inactive 30S subunits about as well as to activated 30S subunits, photolysis of the inactive 30S.probe complex leads to a very different pattern of protein labeling, providing strong evidence, at the protein level, that the inactive to activated transition is accompanied by conformational change in the 1400 region of 16S rRNA.

Autoradiography↗
PubMed1988

[3H]-p-azidopuromycin photoaffinity labeling of Escherichia coli ribosomes: evidence for site-specific interaction at U-2504 and G-2502 in domain V of 23S ribosomal RNA.

Previously we (1) showed that [3H]-p-azidopuromycin was a functional puromycin analogue that, on photolysis in the presence of 70S ribosomes from Escherichia coli, photoincorporated site specifically into proteins L23, L18/22, and L15 [Nicholson, A.W., Hall, C.C., Strycharz, W.A., & Cooperman, B.S. (1982) Biochemistry 21, 3809-3817] and (2) used immunoelectron microscopy to localize the principal sites of p-azidopuromycin photoincorporation within the 50S subunit [Olson, H.M., Nicholson, A.W., Cooperman, B.S., & Glitz, D.G. (1985) J. Biol. Chem. 260, 10326-10331]. These studies are here continued by identification of the principal sites of [3H]-p-azidopuromycin photoincorporation into ribosomal RNA. The major portion of such photoincorporation, 72%, takes place into 23S rRNA. Analysis by hybridization of the photoaffinity-labeled rRNA to restriction enzyme fragments of plasmid pKK3535, which contains rrnB DNA, using a refinement of a recently developed methodology [Hall, C.C., Smith, J.E., & Cooperman, B.S. (1985) Biochemistry 24, 5702-5711], shows that the most prominent [3H]-p-azidopuromycin photoincorporation occurs within bases 2445-2668 in domain V [Noller, H.F. (1984) Annu. Rev. Biochem. 53, 119-162] of 23S rRNA. Photoincorporation into this region is site specific, as demonstrated by the decrease in photoincorporation of radioactivity when unlabeled puromycin is included in the photolysis solution. Significant site-specific photoincorporation also occurs within bases 489-681 in domain II of 23S RNA. Further localization, by the method of reverse transcriptase primer extension [Barta, A., Steiner, G., Brosius, J., Noller, H.F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611], provides evidence that U-2504 and G-2502 are the principal sites of p-azidopuromycin interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed2006

The NMR structure of an internal loop from 23S ribosomal RNA differs from its structure in crystals of 50s ribosomal subunits.

Internal loops play an important role in structure and folding of RNA and in recognition of RNA by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5' 1009CUAAG1013 3'/3' 1168GAAGC1164 5' and 5' 998CUAAG1002 3'/3' 1157GAAGC1153 5' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex 5' 1GGCUAAGAC9 3'/3' 18CCGAAGCUG10 5'. The internal loop forms a different structure in solution and in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.

Deinococcus↗
PubMed2003

Action spectra for UV-light induced RNA-RNA crosslinking in 16S ribosomal RNA in the ribosome.

UV irradiation induces intramolecular crosslinks in ribosomal RNA in the ribosome. These crosslinks occur between nucleotides distant in primary sequence and they are specific, limited in number and have crosslinking efficiencies sufficient to allow their use in monitoring conformational changes. In this work, the frequency of crosslinking for eight 16S rRNA crosslinks was determined as a function of wavelength of irradiation. For six of the crosslinks, the action spectra correspond to the absorption spectra of at least one of the participating nucleotides. For a crosslink between nucleotides C967 and C1400 the maximum frequency of crosslinking occurs at wavelengths blue-shifted from the absorbance maximum of cytidine and for a crosslink between C1402 and C1501 the maximum frequency of crosslinking is red-shifted. Photoreversal of the crosslinks was also studied by deproteinizing crosslinked RNA under mild conditions and then re-irradiating it with specific wavelengths under conditions in which the crosslinks were reversed but not formed. The different crosslinks exhibit significantly different extents of photoreversal versus wavelength profiles. The differences in the crosslinking action spectra can be accounted for in the absorbance spectra of the nucleotides that are involved in the crosslink as well as by the photoreversal action spectra.

Base Sequence↗
PubMed1974

The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites.

With a stepwise degradation and terminal labeling procedure the 3'-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-A(OH). It is suggested that this region of the RNA is able to interact with mRNA and that the 3'-terminal U-U-A(OH) is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA.30S subunit complex.

Bacterial Proteins↗
PubMed1986

Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association.

A nine-base DNA oligomer [d(GTATCTAAT)] was used to probe the accessibility and function of bases in the region 787-795 of Escherichia coli 16S rRNA. Hybridization of the cDNA [d(GTATCTAAT)] to 16S rRNA in situ was carried out by binding the probe to intact 30S ribosomal subunits. Nitrocellulose filter binding showed that cDNA hybridization saturated with increasing probe concentration, suggesting that the probe was binding to a discrete site or sites. RNase H digestion of the rRNA under the DNA . rRNA hybrid and sequencing of the resultant RNA fragments verified that the cDNA probe bound specifically to the 787-795 region. Hybridization experiments using the cDNA probe showed that bases in the 787-795 region of 16S rRNA are exposed on the surface of 30S subunits. The functional role of bases 787-795 was then tested by assaying various ribosomal activities with the cDNA in place. Results of these functional assays demonstrate that this 16S rRNA region is directly involved in the association of 30S and 50S subunits.

Base Sequence↗
PubMed1993

Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site.

From genetic and biochemical evidence, we previously proposed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops. Here, we use "toeprint" experiments with Moloney murine leukemia virus reverse transcriptase to analyze the effect of S15 on the formation of the ternary mRNA-30S-tRNA(fMet) complex. We show that the binding of the 30S subunit on the mRNA stops reverse transcriptase near position +10, corresponding to the 3' terminus of the pseudoknot, most likely by stabilizing the pseudoknot conformation. Furthermore, S15 is found to stabilize the binary 30S-mRNA complex. When the ternary 30S-mRNA-tRNA(fMet) complex is formed, a toeprint is observed at position +17. This toeprint progressively disappears when the ternary complex is formed in the presence of increasing concentrations of S15, while a shift from position +17 to position +10 is observed. Beside, RNase T1 footprinting experiments reveal the simultaneous binding of S15 and 30S subunit on the mRNA. Otherwise, we show by filter binding assays that initiator tRNA remains bound to the 30S subunit even in the presence of S15. Our results indicate that S15 prevents the formation of a functional ternary 30S-mRNA-tRNA(fMet) complex, the ribosome being trapped in a preternary 30S-mRNA-tRNA(fMet) complex.

Base Sequence↗
PubMed1999

Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in escherichia coli carrying temperature-sensitive ribosome recycling factor.

We have isolated a protein, mature RRFHCP, from chloroplasts of spinach (Spinacia oleracea L.) that shows 46% sequence identity and 66% sequence homology with ribosome recycling factor (RRF) of Escherichia coli. RRF recycles ribosomes through disassembly of the posttermination complex. From the cDNA analysis and from the amino-terminal sequencing of the isolated protein, the mature RRFHCP was deduced to have a Mr of 21,838 with 193 aa. It lacks the 78-aa chloroplast targeting sequence encoded by the RRFHCP cDNA sequence. The RRFHCP synthesized in vitro was imported into isolated chloroplasts with simultaneous conversion to the mature RRFHCP. Transcription of the gene coding for RRFHCP was not dependent on light, yet it was limited mostly to photosynthetic tissues in which only one transcript size was detected. Mature RRFHCP exerted a bactericidal effect on E. coli carrying temperature-sensitive RRF at the permissive temperature whereas wild-type E. coli was not affected.

Amino Acid Sequence↗
PubMed1997

Binding of mammalian ribosomal protein complex P0.P1.P2 and protein L12 to the GTPase-associated domain of 28 S ribosomal RNA and effect on the accessibility to anti-28 S RNA autoantibody.

We have investigated binding of rat ribosomal proteins to the "GTPase domain" of 28 S rRNA and its effect on accessibility to the anti-28 S autoantibody, which recognizes a unique tertiary structure of this RNA domain. Ribosomal protein L12 and P protein complex (P complex) consisting of P0, P1, and P2 both bound to the GTPase domain of rat 28 S rRNA in a buffer containing Mg2. Chemical footprinting analysis of their binding sites revealed that the P complex mainly protected a conserved internal loop region comprising residues 1855-1861 and 1920-1922, whereas L12 protected an adjacent helix region encompassing residues 1867-1878 and 1887-1899. These sites are close to but distinct from the binding site for anti-28 S antibody determined previously. The bindings of P complex and L12 increased the anti-28 S accessibility, as revealed by gel retardation and quantitative immunoprecipitation analyses. In a Mg2+-eliminated condition, the RNA failed to bind to either anti-28 S or L12 but assembled into a complex under their coexistence. However, the RNA retained a property of binding to the P complex even in the absence of Mg2+, and this binding conferred high anti-28 S accessibility. These results indicated that the bindings of the P complex and L12 to their respective sites influenced the GTPase domain to increase the accessibility to anti-28 S. A possible RNA conformation adjusted by the protein bindings is discussed.

Animals↗
PubMed1999

Effects of tetracycline and spectinomycin on the tertiary structure of ribosomal RNA in the Escherichia coli 30 S ribosomal subunit.

Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967xC1400 completely, increases the frequency of cross-link C1402x1501 twofold, and decreases the cross-link G894xU244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934xU1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected cross-linking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect.

Anti-Bacterial Agents↗
PubMed2004

Ribosomal RNA processing and ribosome biogenesis in eukaryotes.

In eukaryotes nearly 500 rRNAs, ribosomal proteins, snoRNAs and trans-acting factors contribute to ribosome biogenesis. After more than 30 years of intense research, the incredible complexities of nucleolar function are revealed but details often remain unclear. Here we review this progress and the many intriguing questions which remain.

Active Transport, Cell Nucleus↗
PubMed2000

In vivo release of mitotic silencing of ribosomal gene transcription does not give rise to precursor ribosomal RNA processing.

Nuclear RNA transcription is repressed when eukaryotic cells enter mitosis. Here, we found that the derepression of ribosomal gene (rDNA) transcription that normally takes place in telophase may be induced in prometaphase, metaphase, and anaphase mitotic HeLa cells, and therefore appears not to be dependent on completion of mitosis. We demonstrate for the first time that in vivo inhibition of the cdc2- cyclin B kinase activity is sufficient to give rise to okadaic acid-sensitive dephosphorylation of the mitotically phosphorylated forms of components of the rDNA transcription machinery, and consequently to restore rDNA transcription in mitotic cells. These results, showing that during mitosis the rDNA transcription machinery is maintained repressed by the cdc2-cyclin B kinase activity, provide an in vivo demonstration of the cell cycle-dependent regulation of rDNA transcription. Interestingly in mitotic cells, the newly synthesized 47S precursor ribosomal RNA (pre-rRNA) is not processed into the mature rRNAs, indicating that rDNA transcription and pre-rRNA processing may be uncoupled. Moreover this suggests that inhibition of the cdc2- cyclin B kinase is not sufficient to activate the 47S pre-rRNA processing machinery and/or to induce its relocalization at the level of newly synthesized 47S pre-rRNA. This in vivo approach provides new possibilities to investigate the correlation between pre-rRNA synthesis and pre-rRNA processing when the nucleolus reforms.

CDC2 Protein Kinase↗
PubMed2000

Crystal structure of ribosomal protein L4 shows RNA-binding sites for ribosome incorporation and feedback control of the S10 operon.

Ribosomal protein L4 resides near the peptidyl transferase center of the bacterial ribosome and may, together with rRNA and proteins L2 and L3, actively participate in the catalysis of peptide bond formation. Escherichia coli L4 is also an autogenous feedback regulator of transcription and translation of the 11 gene S10 operon. The crystal structure of L4 from Thermotoga maritima at 1.7 A resolution shows the protein with an alternating alpha/beta fold and a large disordered loop region. Two separate binding sites for RNA are discernible. The N-terminal site, responsible for binding to rRNA, consists of the disordered loop with flanking alpha-helices. The C-terminal site, a prime candidate for the interaction with the leader sequence of the S10 mRNA, involves two non-consecutive alpha-helices. The structure also suggests a C-terminal protein-binding interface, through which L4 could be interacting with protein components of the transcriptional and/or translational machineries.

Amino Acid Sequence↗
PubMed1989

Ribosomal proteins S7 and L1 are located close to the decoding site of E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3'-end of the anticodon.

Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.

Affinity Labels↗
PubMed1989

Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site.

Three different mRNA analogues (28 to 34 nucleotides long) were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 5'-side of these coding triplets. A photo-reactive group was introduced by substitution of the thio-U with 4-azidophenacyl bromide. The messages were bound to E. coli 70S ribosomes in the presence of the appropriate tRNA-Thr or tRNA-Ala, and the azidophenyl group was photoactivated. Cross-linking was found to occur exclusively within the 30S subunit, with the 32P-label in the cross-linked mRNA being divided roughly equally between 30S ribosomal proteins and 16S RNA. Immunological analysis of the cross-linked proteins showed that, in the presence of either tRNA species, protein S7 was the primary target, whereas in the absence of tRNA only small amounts of protein S21 were cross-linked. The cross-link site to 16S RNA lay in all cases very close to its extreme 3'-terminus. These data indicate that the outgoing message leaves the cleft of the 30S subunit in a "northerly" direction.

Base Sequence↗
PubMed1975

The secondary structure of E. coli ribosomes and ribosomal RNA's: a spectrophotometric approach.

The thermal denaturation spectra of E. coli 50s and 30s ribosomal sub units and of their isolated RNA's were studied over the wavelength range of 2300-3000 Angstrom. It was possible to fit the experimental denaturation spectra with super positions of the reference spectra for denaturation of A - U and G - C base pairs derived from model polyribonucleotides. The coefficients of these linear combinations were used to calculate the fractions of A - U and G - C base pairs in the samples. It was found that the helical content of the RNA's inside the subunits is smaller than that of the isolated RNA's, thus suggesting that proteins may affect the secondary structure of RNA in the ribosome.

Escherichia↗
PubMed2001

Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA.

Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

Base Sequence↗