Ribosomal protein S18e is a structural constituent of the 40S ribosomal subunit. We obtained recombinant human ribosomal protein S18e and studied its structural and functional properties. With the ...use of CD spectroscopy we showed that the protein secondary structure is mainly helical and stable in the neutral pH range and at low urea concentrations. Applying multiple sequence alignment, we revealed that the protein structure has characteristics of the eukaryotic members of the ribosomal protein S13p family with additional extensions in the N-terminal and central parts that contain α-helices according to our prediction. S18e binds specifically and independently to an RNA transcript corresponding to the evolutionary core of the 3′-major domain of 18S rRNA. Hydroxyl radical footprinting showed that the binding site of S18e on the 18S rRNA is similar in general to the binding site of S13p on the 16S rRNA in the 30S ribosomal subunit, albeit the rRNA regions attributed to binding of the eukaryote-specific extensions of S18e were also detected. With magnesium ion concentration close to cellular conditions (2
mM), protein binding caused substantial rearrangements in the rRNA transcript making it compact in such a manner that helices H29/H30 and H41–H43 form a bundle resembling their arrangement in the ribosome. Thus, S18e seems to act as a molecular staple fixing the 18S rRNA 3′-major domain core.
► S18e binds specifically to 18S rRNA without assistance from other ribosomal proteins. ► S18e fixes the structure of the 18S rRNA 3′-major domain core acting as a staple. ► The eukaryote-specific extensions of S18e are involved in binding to the 18S rRNA. ► S18e is capable of folding the rRNA structure under a low Mg
2+ concentration.
Human ribosomal proteins S5e and S16e are the homologues of prokaryotic S7p and S9p, respectively. It was shown that S5e and S16e are capable of the specific binding with a rRNA transcript ...corresponding to the region of human 18S rRNA containing helices H28–30 and H41–43 (3Dm), which is homologous to the region in 16S rRNA containing the entire binding site for S7p and the major part of the site for S9p. We have studied binding of S5e and S16e to 3Dm and demonstrated that while each of them is able to bind to the rRNA transcript independently, their simultaneous binding has a noticeable synergetic effect. Using enzymatic footprinting, we showed that these proteins protect 3Dm against hydrolysis with RNases mainly in the regions homologous to the sites of S7p and S9p binding on the 16S rRNA. At the same time, we found regions that correspond to 16S rRNA fragments distant from the binding sites of the respective homologous prokaryotic proteins. Comparison of these results with the data on 3Dm footprinting in binary complexes with S5e or S16e revealed that each of these proteins affects binding of another one to 3Dm, which is displayed in significant expansion of 3Dm sites protected by the proteins against hydrolysis in the ternary complex.
Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at ...the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.
To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. ...Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3′-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA
Phe, targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3′-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.
This article is devoted to the social vulnerability of freelance workers and the way it is represented in different dimensions of precariousness. According to the previous studies, we identifed the ...categories of social insurance (fnancial, juridical, lack of social guaranteesand lack of personal well-being) and the most common indicators within each. We conducted a qualitative research using in-depth semi-formal interviews with 21 employees older than 18 years, who are engaged in freelancing at the moment or had such experience in the last couple of years. The number of informants included employees for whom freelance is one of the main sources of income for at least one year. During the interview, the informants were speaking about the subjective assessment of social precariousness and also answered to some questions aimed at identifying the objective features of the precious situation in employment status. In the analysis of subjective assessments of social precariousness, it was revealed that informants cannot be divided into categories according to the degree of precariousness, because they can experience social vulnerability in one or several areas at the same time. According to the results of the study, we propose to consider precariousness as a certain scale from 0 to the maximum value of the insecurity parameters. This approach will take into account the importance of subjective assessment of freelancers’ position, while the dichotomy “precariat — free agent”, which is mostly shared by researchers, does not consider the complexity of this social phenomenon.
The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by ...mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.
Human recombinant ribosomal protein S26 (rpS26) was shown to interact with its pre-mRNA intron I and mRNA fragment. Endogenous rpS26 in HeLa nuclear extract was also found to bind to the intron I, ...and with a lower extent to the mRNA fragment. The addition of recombinant rpS26 to the nuclear extract increased the binding largely. The in vitro splicing of an RNA that contained exon I, intron I and part of exon II of the rpS26 pre-mRNA yielded conventional and alternative mRNAs. Recombinant rpS26 was found to suppress the formation of both mRNAs. Sites of the pre-mRNA involved in the binding to rpS26 were detected by toe-printing. Nucleotides that caused a stop (pause) of the reverse transcription formed two clusters on the RNA secondary structure. One cluster including A69, A287 and A303 arranged the conventional 3′ site of splicing, another one including A131, A136, G156, A166 and A264 arranged the alternative 3′ site of splicing.
Positioning of the mRNA codon towards the 18S ribosomal RNA in the A site of human 80S ribosomes has been studied applying short mRNA analogs containing either the stop codon UAA or the sense codon ...UCA with a perfluoroaryl azide group at the uridine residue. Bound to the ribosomal A site, a modified codon crosslinks exclusively to the 40S subunits under mild UV irradiation. This result is inconsistent with the hypothesis Ivanov et al. (2001) RNA 7, 1683–1692 which requires direct contact between the large rRNA and the stop codon of the mRNA as recognition step at translation termination. Both sense and stop codons crosslink to the same A1823/A1824 invariant dinucleotide in helix 44 of 18S rRNA. The data point to the resemblance between the ternary complexes formed at elongation (sense codon·aminoacyl-tRNA·AA dinucleotide of 18S rRNA) and termination (stop codon·eRF1·AA dinucleotide of 18S rRNA) steps of protein synthesis and support the view that eRF1 may be considered as a functional mimic of aminoacyl-tRNA.
The protein components of human 40S ribosomal subunits were dissociated by centrifugation in gradients of sucrose and LiCl in the presence of 0.5 M KCl. The proteins that split off were analyzed by ...SDS-PAGE and 2D-PAGE. The order of dissociation of the proteins, depending on the salt concentration (from 0.8 M to 1.55 M), was established. The majority of the proteins started to split off simultaneously at a monovalent cation concentration of 0.8 M. Ten proteins were found to be more resistant; of these proteins S7, S10, S16, and S19 were retained most strongly and thereby may be considered to be core proteins.
The interaction between mRNA and 18S rRNA in human 80S ribosomes has been studied using synthetic mRNA analogues randomly substituted with 4-thiouridine, which can be photoactivated for ...cross-linking. Two mRNA analogues with different sequences have been used for complex formation with ribosomes without or with the presence of a cognate tRNA. Cross-linked 18S rRNA nucleotides were identified by reverse transcription analysis. The base U630 in 18S rRNA was the main target of cross-linking for both of the mRNA analogues studied, and three minor sites of cross-linking, A1060, U1046, and U966, were also identified. Thus, in the case of human 80S ribosomes, the set of nucleotide residues cross-linked to the mRNA analogues is significantly smaller than the twelve sites seen for Escherichia coli with these same two mRNA analogues Bhangu, R., & Wollenzien, P. (1992) Biochemistry 31, 5937-5944. The residue U630 is within a highly conserved region corresponding to the 530 loop region of eubacterial 16S rRNA; the cross-link to this site indicates that it plays a key role in interacting with mRNA on 80S ribosomes independently of the presence of a cognate tRNA at the P site.
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