Ribosomal Protein Binding Sites Research Articles

Structural Distinctive 26SK, a Ribosome-Inactivating Protein from Jatropha curcas and Its Biological Activities.

Ribosome-inactivating proteins (RIPs) are a group of proteins exhibiting N-glycosidase activity leading to an inactivation of protein synthesis. Thirteen predicted Jatropha curcas RIP sequences could be grouped into RIP types 1 or 2. The expression of the RIP genes was detected in seed kernels, seed coats, and leaves. The full-length cDNA of two RIP genes (26SK and 34.7(A)SK) were cloned and studied. The 34.7(A)SK protein was successfully expressed in the host cells while it was difficult to produce even only a small amount of the 26SK protein. Therefore, the crude proteins were used from E. coli expressing 26SK and 34.7(A)SK constructs and they showed RIP activity. Only the cell lysate from 26SK could inhibit the growth of E. coli. In addition, the crude protein extracted from 26SK expressing cells displayed the effect on the growth of MDA-MB-231, a human breast cancer cell line. Based on in silico analysis, all 13 J. curcas RIPs contained RNA and ribosomal P2 stalk protein binding sites; however, the C-terminal region of the P2 stalk binding site was lacking in the 26SK structure. In addition, an amphipathic distribution between positive and negative potential was observed only in the 26SK protein, similar to that found in the anti-microbial peptide. These findings suggested that this 26SK protein structure might have contributed to its toxicity, suggesting potential uses against pathogenic bacteria in the future.

The role of mRNA structure in bacterial translational regulation.

The characteristics of bacterial messenger RNAs (mRNAs) that influence translation efficiency provide many convenient handles for regulation of gene expression, especially when coupled with the processes of transcription termination and mRNA degradation. An mRNA's structure, especially near the site of initiation, has profound consequences for how readily it is translated. This property allows bacterial gene expression to be altered by changes to mRNA structure induced by temperature, or interactions with a wide variety of cellular components including small molecules, other RNAs (such as sRNAs and tRNAs), and RNA-binding proteins. This review discusses the links between mRNA structure and translation efficiency, and how mRNA structure is manipulated by conditions and signals within the cell to regulate gene expression. The range of RNA regulators discussed follows a continuum from very complex tertiary structures such as riboswitch aptamers and ribosomal protein-binding sites to thermosensors and mRNA:sRNA interactions that involve only base-pairing interactions. Furthermore, the high degrees of diversity observed for both mRNA structures and the mechanisms by which inhibition of translation occur have significant consequences for understanding the evolution of bacterial translational regulation. WIREs RNA 2017, 8:e1370. doi: 10.1002/wrna.1370 For further resources related to this article, please visit the WIREs website.

Reflections on the 20th anniversary of RNA.

Reflections on the 20th anniversary of RNA.

Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome's structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, we have recently shown that an active hybrid ribosome whose 16S rRNA has been specifically substituted with that from non-E. coli bacteria can be reconstituted in vivo. To investigate the mutational robustness of 16S rRNA and the structural basis for its functionality, we used a metagenomic approach to screen for 16S rRNA genes that complement the growth of E. coli Δ7. Various functional genes were obtained from the Gammaproteobacteria and Betaproteobacteria lineages. Despite the large sequence diversity (80.9-99.0% identity with E. coli 16S rRNA) of the functional 16S rRNA molecules, the doubling times (DTs) of each mutant increased only modestly with decreasing sequence identity (average increase in DT, 4.6 s per mutation). The three-dimensional structure of the 30S ribosome showed that at least 40.7% (628/1,542) of the nucleotides were variable, even at ribosomal protein-binding sites, provided that the secondary structures were properly conserved. Our results clearly demonstrate that 16S rRNA functionality largely depends on the secondary structure but not on the sequence itself.

Cloning, Sequencing, and Characterization of Ribosomal Protein and RNA Polymerase Genes from the Region Analogous to the α-Operon of Escherichia coli in Halophilic Archaea, Halobacterium halobium

A determination was made of the nucleotide sequence of the 3215-bp region of a ribosomal protein gene cluster (HS13, HS4, HS11, and HeL18), RNA polymerase (RNA poly D), and tRNA genes (tRNAser and tRNAarg) of halophilic Archaea Halobacterium halobium, which is analogous to the α-operon of Escherichia coli (tRNAser-HS13-HS4-HS11-RNA poly D-tRNAarg-HeL18). The seven-gene string was preceded by a pseudoknot-like structure similar to the proposed S4 ribosomal protein binding site of the α-operon mRNA leader in E. coli. Using an inducible expression system H. halobium HS4 was produced in large amounts in E. coli, and immunoblot analysis showed the S4 to constitute a 21-kDa polypeptide component of the ribosome. Analysis of the deduced amino acids sequence revealed that the HS13, HS4, and HS11 sequences including the RNA polymerase subunit are more similar to their eukaryotic than to their bacterial counterparts. HeL18, located downstream of the gene cluster analogous to the E. coli α-operon (S13-S11-S4-RNA poly D-L17), was similar to both the eukaryotic (eL18) and eubacterial ribosomal protein L15 located in the spc-operon, but not to L17 positioned as the terminal gene of the bacterial α-operon.

Analysis of the Ribosome Large Subunit Assembly and 23 S rRNA Stability in Vivo

The ability of mutant 23 S ribosomal RNA to form particles with proteins of the large ribosomal subunit in vivowas studied. A series of overlapping deletions covering the entire 23 S rRNA, were constructed in the plasmid copy of an E. coli23 S rRNA gene. The mutant genes were expressed in vivousing an inducible tacpromoter. Mutant species of 23 S rRNA, containing deletions between positions 40 and 2773, were incorporated into stable ribonucleoprotein particles. In contrast, if one end of the 23 S rRNA was deleted, the mutant rRNA was unstable and did not form ribosomal particles. Protein composition of the mutant particles was specific; the presence of the primary rRNA-binding proteins corresponded to their known binding sites. Furthermore, several previously unknown ribosomal protein binding sites in 23 S rRNA were identified. Implications of the results on ribosome assembly are discussed.

Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling*1

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1), with a carbodiimide derivative (at G(N-1) and U(N-3)) and with kethoxal (at G(N-1, N-2)). Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7)) and dimethylsulfate (at G(N-7)). Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A·G, G·U, G·G, A·C, C·C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.

Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1), with a carbodiimide derivative (at G(N-1) and U(N-3)) and with kethoxal (at G(N-1, N-2)). Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7)) and dimethylsulfate (at G(N-7)). Double-stranded or stacked regions were tested with RNase V 1 and unpaired guanine residues with RNase T 1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A·G, G·U, G·G, A·C, C·C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.

Ribosomal proteins EL11 from Escherichia coli and L15 from Saccharomyces cerevisiae bind to the same site in both yeast 26 S and mouse 28 S rRNA

The heterologous interaction of Escherichia coli ribosomal protein EL11 with yeast 26 S and mouse 28 S rRNA was studied by analysing the ability of this protein to form a specific complex with various synthetic rRNA fragments that span the structural equivalent of the EL11 binding site present in these eukaryotic rRNAs. The fragments were obtained by SP6 polymerase-directed in-vitro run-off transcription of parts of the yeast or mouse large rRNA gene cloned behind the SP6 promoter. EL11 was found to protect an oligonucleotide fragment of 63 nucleotides from both the yeast and mouse transcripts against digestion by RNase T 1. In both cases, the position of this fragment in the L-rRNA § § Abbreviation used: L-rRNA, high-molecular-weight rRNA present in the large ribosomal subunit; r-protein, ribosomal protein. sequence coincides almost exactly with that of the fragment previously found to be protected by EL11 in E. coli 23 S rRNA. Moreover, the protected yeast fragment was shown to be able to re-bind to EL11 by a nitrocellulose filter binding assay. A ribosomal protein preparation from Saccharomyces cerevisiae containing L15 (YL23) as well as the acidic proteins L44′, L44 and L45 protects exactly the same oligonucleotide fragment as does EL11 in both the yeast and mouse transcripts. Evidence is provided that L15, which is known to be structurally and functionally equivalent to EL11, is the rRNA-binding protein in this preparation. Thus the structural equivalent of the EL11 binding site present in yeast 26 S rRNA constitutes the second example of functional conservation of a ribosomal protein-binding site on rRNA between prokaryotes and eukaryotes.

Construction and Functional Analysis of Ribosomal 5S RNA fromEscherichia coliwith Single Base Changes in the Ribosomal Protein Binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.

Translational regulation of the L11 ribosomal protein operon of Escherichia coli: analysis of the mRNA target site using oligonucleotide-directed mutagenesis.

The L11 ribosomal protein operon in Escherichia coli consists of the genes for proteins L11 and L1 and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the translation initiation site of the first L11 cistron. Several mutant plasmid molecules carrying altered nucleotide sequences in the L1 target site were constructed by site-directed in vitro mutagenesis using synthetic oligodeoxyribonucleotides. Specifically, we examined the importance of a presumptive double-stranded stem structure that is common among L1 binding sites on rRNA from a variety of organisms and in L11 mRNA. Mutational alterations that disrupt the stem structure were found to abolish translational regulation as analyzed both in vitro and in vivo. Two of the mutations were combined so that the stem structure was restored but with a different primary nucleotide sequence. This double mutant was shown to restore the original phenotype, the ability to be translationally regulated by L1. These experiments show the importance of the stem structure, but not its primary sequence, for the interaction of L1 with the mRNA and support the concept that mRNA target sites share some structural features with the corresponding ribosomal protein binding sites of rRNA.

The ribosomal protein binding site in Saccharomyces cerevisiae ribosomal 5 S RNA. A conserved protein binding site in 5 S RNA.

The ribosomal protein binding site in Saccharomyces cerevisiae ribosomal 5 S RNA. A conserved protein binding site in 5 S RNA.

Conservation of ribosomal protein binding sites in prokaryotic 16S RNAs.

The Escherichia coli 30S ribosomal subunit proteins S4, S7, S8, S15, S17, and S20 that interact independently with 16S RNA from E. coli formed specific heterologous complexes with 16S RNAs extracted from 11 different prokaryotes covering a broad phylogenetic range. Complex formation was shown to be specific by saturation of binding in the presence of excess protein. Binding stoichiometries and the apparent affinities for a given protein varied depending on which 16S RNA was used, although the pattern of binding was not strictly correlated with phylogenetic relationships. The size-distribution of fragments resulting from limited hydrolysis of free prokaryotic 16S RNAs with T1 and pancreatic ribonucleases indicated that the structural organization of 16S RNA from E. coli is similar to that of 16S RNAs from closely related species, but differs, although to an unknown extent, from that of 16S RNAs from other prokaryotes tested. Digestions of RNA-protein complexes under similar conditions indicated that the proteins remain bound to specific RNA fragments. For those 16S RNAs isolated from species closely related to E. coli, the fragments were comparable to those generated by hydrolysis of the homologous complex.

RNA-protein interactions in the ribosome: III. Conformation and stability of ribosomal protein binding sites in the 16 S RNA

Following dialysis against distilled water, the 16 S ribosomal RNA of Escherichia coli is unable to interact with 30 S subunit protein S4 at 0 °C. The dialysed RNA recovered this capacity, however, when heated at 40 °C in the presence of 0.02 m-MgCl 2 prior to addition of the protein. Furthermore, its sensitivity to ribo-nuclease markedly declined and its sedimentation rate increased as a consequence of this treatment. Although no concomitant changes in secondary structure were detected by absorbance and fluorescence techniques, the rearrangement of a small number of base-pairs was not excluded. Kinetic measurements revealed that binding site reactivation satisfies the first-order rate law and that the process is highly temperature-dependent, exhibiting an Arrhenius activation energy of 40,800 cal/mol. Together, these data suggest that dialysed RNA undergoes a unimolecular conformational transition upon pre-incubation in Mg 2+-containing buffers and that this transition leads to renaturation of the binding site for protein S4. Similar results were obtained for several other proteins of the 30 S subunit. In particular, S7, S16/S17 and S20 all failed to interact efficiently with dialysed 16 S RNA at 0 °C. These proteins bound normally to the RNA, however, after it had been incubated at 40 °C in the presence of Mg 2+ ions. By contrast, prior dialysis of the 16 S RNA did not affect its ability to associate with S8 and S15 at 0 °C. These two proteins interacted equally well with dialysed and pre-incubated 16 S RNA, indicating that their binding sites are not susceptible to the reversible alterations in conformation which influence the attachment of the other RNA-binding proteins to the nucleic acid molecule. The effects of dialysis and pre-incubation on the interaction of 16 S RNA with an unfractionated mixture of 30 S subunit proteins were also investigated. The dialysed RNA bound only S6, S8, S15 and S18 at 0 °C whereas, after heating at. high Mg 2+ concentrations, the RNA associated with S4, S7, S9, S13, S16/S17, S19 and S20 as well. These results leave little doubt that the protein-binding capacities of the 16 S RNA are intimately related to its three-dimensional configuration, although individual binding sites appear to differ significantly in their stability to small changes in structure.

A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites

A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971). We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence.

Primary sequence of the 16S ribosomal RNA of Escherichia coli.

Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.

Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli.

Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA. The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule. Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.

RNA-protein interactions in the ribosome: I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes

Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the S16 S17 mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of S16 S17 was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T 1 or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and S16 S17 as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.

Location of ribosomal protein binding sites on 16S ribosomal RNA.

The distribution of ribosomal protein binding sites on the 16S ribosomal RNA molecule has been analyzed by limited ribonuclease hydrolysis of RNA-protein complexes, as well as by the interaction of individual proteins with RNA fragments purified from partial enzymatic digests. Of the six 30S subunit proteins known to interact directly with 16S RNA, proteins S4, S8, S15, S20, and, probably, S13 bind within a fragment produced by T(1) RNase (12S RNA) that comprises some 900 nucleotides and covers almost the entire 5'-terminal half of the 16S molecule. A fragment of 500-600 nucleotides (8S RNA) that is contiguous with 12S RNA and arises from the 3'-terminal portion of the 16S molecule is believed to contain the binding site for protein S7. Protein S15 interacts specifically with a sequence of about 135 nucleotides (4S RNA) that derives from 12S RNA after more extensive hydrolysis. Protein S4, but none of the other ribosomal proteins, binds to a 500-nucleotide fragment (9S RNA), generated by pancreatic RNase, that lies at the 5'-terminus of 16S RNA and is completely overlapped by the 12S fragment. A preliminary map of the binding sites is presented.