Requirement of chain initiation factor 3 and ribosomal protein S1 in translation of synthetic and natural messenger RNA.

Amino acid incorporation directed by poly(A), poly(U) or R17 RNA has been examined in S1-depleted protein synthesizing systems. We observe that the translation of either synthetic or natural messenger RNA is strictly dependent on the presence of chain initiation factor 3 and ribosomal protein S1. With poly(A) or poly(U) both IF-3 and S1 stimulate amino acid incorporation at least 25-fold, and with R17 RNA the stimulation is approximately 15-fold. More than one copy of S1 per ribosome decreases amino acid incorporation directed by poly(U) or R17 RNA. Initiation complex formation with R17 RNA is also stimulated optimally by the addition of one copy of S1 per ribosome. The function of IF-3 and S1 in protein synthesis is considered.     

Functional heterogeneity of the 30S ribosomal subunit of E. coli

Summary When 30S ribosomal subunits from E. coli are incubated with poly U, two separable components are recovered by zonal centrifugation of the incubation mixture. The faster sedimenting component is an aggregate of 30S subunits and poly U, while the slower one corresponds to the 30S ribosomal subunit. One ribosomal protein, protein 30S-1 is predominantly present in the faster sedimenting aggregate. The amount of poly U-30S subunit complex formed in the incubation mixture is limited by the amount of protein 30S-1 present. Consequently the number of ribosomal binding sites available for Phe-tRNA is limited in a similar fashion by the presence of protein 30S-1. When 30S ribosomal subunits are reconstituted in the absence of protein 30S-1, very little poly U or Phe-tRNA binding capacity is manifest under our assay conditions. We conclude that protein 30S-1 is required for maximum capacity of ribosomes to bind mRNA. Since this protein is present only on a fraction of the ribosome at any one time, it must exchange from one ribosome to another during protein synthesis.Abbreviations Poly U (polyuridylic acid) - t-RNA (transfer ribonucleic acid) - mRNA (messenger ribonucleic acid) - Phe (phenylanine) - A₂₆₀ unit (unit of material which gives an optical density of 1.0 at 260 nm in a one centimeter optical path)     

On the role of protein S4 N-terminal residues 1 through 30 in 30S ribosome function.

30S ribosomal protein S4 contains a single cysteine residue at position 31. We have selectively cleaved the peptide bond adjacent to this residue using the reagent 2-nitro-5-thiocyanobenzoic acid. The two resultant fragments were purified. The smaller S4-fragment (1-30) was found to be incapable of interacting with 16S RNA directly. This fragment also is not incorporated into a particle reconstituted from 16S RNA and 20 purified proteins with S4 missing. In contrast, the large S4-fragment (31-203) appears to be fully functional in ribosome assembly. Replacement of S4 with this fragment in the reconstitution reaction leads to a complete 30S ribosome containing all 30S proteins. This particle has a full capacity to bind poly U but has lost all activity for poly U directed phe-tRNA binding. We therefore propose that the N-terminus of protein S4 is not critical for ribosome assembly but is essential for tRNA binding.     

Spatial organization of template polynucleotides on the ribosome determined by fluorescence methods.

The spatial organization of template polynucleotides on the ribosome and the dynamics of their interaction with 30 S subunits have been studied by fluorescence spectroscopy. The topography of the mRNA in the ribosome has been determined using singlet-singlet energy transfer. This method has allowed us to estimate distances between donors and acceptors of energy which have been linked to the terminal residues of template polynucleotides (poly- and oligo(U) and oligo(A] and 16 S RNA or to SH-groups of ribosomal proteins S1 and S8. The dynamics of mRNA-ribosome interaction have been investigated by the fluorescence stopped-flow technique. It has been shown that the binding to the 30 S subunit of poly(U) with length much shorter (16 nucleotides) than that covered by the ribosome is greatly enhanced by protein S1. However, the final position of oligo(U)16 on the 30 S subunit, which probably includes the ribosomal decoding site, proves to be quite different from that occupied by oligo(U)16 on a free protein S1. Interaction of oligo- and poly(U) with the 30 S subunit occurs in at least two steps: the first one is as fast as the interaction of poly(U) with free S1, whereas the second step represents a first-order reaction. Therefore, the second step may reflect some rearrangement of the template in the ribosome after its primary binding. It is suggested that protein S1 in some cases may fulfill the role of a transient binding site for mRNA in the course of its interaction with the ribosome. The general shape of the template in the mRNA binding region of the ribosome has been studied using various synthetic ribopolynucleotides and has been shown to be similar. It can be represented by a loop(s) or "U-turn(s)". On the basis of estimation of distances from the ends of poly(U) to some well-localized points on the 30 S ribosomal surface, a tentative model of mRNA path through the ribosome is proposed.     

Evidence that proteins S1, S11 and S21 directly participates in the binding of transfer RNA to the 30S ribosome.

In a previous publication1 we reported that the tyrosine selective reagent, tetraitromethane, causes complete inactivation of E. coli 30S ribosomes for poly U directed non-enzymatic phe-tRNA binding. This inactivation was demonstrated to be due to the chemical modification of the protein moiety of the ribosome. We have no identified the proteins of the 30S particle inactivated by this modification. Using a method of ribosome reconstruction we have found that unmodified proteins S1, S11, and S21 are essential for the restoration of the phe-tRNA binding activity of tetranitromethane inactivated ribosomes. We propose that these three proteins are intimately involved in the 30S ribosome binding site for tRNA.     

Studies on the role of amino acid residues 31 through 46 of ribosomal protein S4 in the mechanism of 30 S ribosome assembly.

We have described previously the isolation of a large fragment of 30 S ribosomal protein S4 (Changchien &; Craven, 1976). This S4-fragment is produced by the digestion of the S4–16S RNA complex with trypsin and it retains a full capacity to associate specifically with 16S RNA. It was also demonstrated that the S4-fragment has approximately 46 amino acid residues missing from the N-terminus and an intact C-terminus (also shown by Newberry et al., 1977). Preliminary experiments with this S4-fragment indicated that it could not fully replace the intact protein S4 in the process of 30 S ribosome assembly in vitro.We have also recently reported (Changchien et al., 1978) the preparation of a new fragment of protein S4 which has only 30 amino acid residues cleaved from the N-terminus. This was achieved by the use of the reagent 2-nitro-5-thiocyanobenzoic acid which selectively modifies the cysteine residue at position 31 followed by a cleavage of the adjacent peptide bond.We have now fully characterized the capacity of these two fragments, S4-fragment (47–203) and S4-fragment(31–203), to participate in the 30 S ribosome assembly process in vitro. Using 2-dimensional polyacrylamide gel electrophoresis, we find that when S4-fragment(47–203) is a component of the in vitro assembly reaction, proteins S1, S2, S10, S18 and S21 fail to become incorporated into the final particle. In contrast, S4-fragment(31–203) appears to participate in the reconstitution reaction without impairment allowing the complete incorporation of all 20 proteins of the 30 S subunit. The resultant particle, containing the S4-fragment (31–203), is fully active in the binding of poly(U), but is completely inactive for non-enzymatic poly(U)-directed binding of Phe-tRNA (Changchien et al., 1978). These results suggest that residues 1 through 30 of protein S4 are not involved in the assembly of the 30 S ribosome, but are required for the proper construction of the tRNA binding site. In addition residues 31 through 46 must be somehow critically important for the assembly of proteins S1, S2, S10, S18 and S21. We present evidence to show that the absence of residues 31 through 46 of protein S4 prevents a conformational change in the structure of 16 S RNA which normally accompanies the RI to RI transition and that this results in the inability of these proteins to participate in the assembly process.     

Comparison by photoaffinity labeling of the proteins involved in GTP hydrolysis from 70S ribosomes and a 5S RNA-protein complex from Bacillus stearothermophilus.

Photoaffinity labeling of 70S ribosomes from B. stearothermophilus by [³H]-1-(4-azidophenyl)-2-(5′-guanyl) pyrophosphate (APh-GDP) in the presence of fusidate and elongation factor G (EF-G) results in incorporation of tritium in the 50S proteins BL2, BL10 and BL22. Irradiation of the corresponding 5S RNA-protein complex in the presence of the GDP derivative gives only incorporation of tritium in BL10 and BL22. The proteins BL10 and BL22 comigrate in two dimensional gel electrophoresis with the 50S ribosomal proteins EL11 and EL18 from E. coli. The result suggests that the region at or near the guanine nucleotide binding site of the ribosome and the complex are the same. Since previous work has shown that the latter two are labeled upon irradiation of the ribosome with [³H]-APh-GDP, it is concluded that ribosomes from E. coli and B. stearothermophilus have structurally related GTPase sites.     

The major ribosome binding site of Escherichia coli ribosomal protein S1 is located in its N-terminal segment

We have cleaved protein S1, which is the largest and the most elongated protein of the Escherichia coli ribosome, using cyanogen bromide and isolated two fragments that retain the functional domains of the intact molecule. The fragments (denoted S1-F2a and S1-F2b) showed molecular weights of 24,000 and 22,500 by dodecyl sulphate/polyacrylamide gel electrophoresis. Fragment F2a is shown to be the N-terminal segment containing about 32% of the peptide chain length of S1. Fragment F2b is derived from another (probably C-terminal) region of S1.Fragment F2a binds to 30 S ribosomal subunits with a strength and specificity comparable to the binding of intact S1. It also binds to matrix-bound poly(U) but the binding is salt-sensitive, unlike the binding of intact S1. Fragment F2b binds only very weakly to poly(U) and does not bind to 30 S subunits. These results are discussed with respect to the ribosome binding domain(s) of protein S1 and the possible interdependence of the multiple functional domains in this large protein.     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.     

Influence of heating on the formation of ribosome couples and 80S initiation complex from the 40S and 60S ribosomal subunits from rabbit reticulocytes*

Summary Poly(U)-and AUG-dependent initiations were studied under conditions in which the ribosomal subunits from rabbit reticulocytes form 80S ribosome couples. The AUG-dependent initiation took place only in the presence of 40S subunits and not in that of 80S ribosome couples. The poly(U)-dependent initiation could take place on 80S ribosome couples as well as on 40S subunits. Heating of 60S subunits removed their inhibitory effect on AUG-dependent initiation without affecting their ability to attach to the 40S-AUG-initiator-tRNA complex and covert it to the more thermostable 40S-AUG-60S complex. This complex could bind initiator-tRNA reversibly. Two components seemed to be present in ribosomal high salt wash which antagonistically caused the disappearance and persistence of the inhibitory effect of 60S subunits on AUG-dependent initiation. Paper No. 3 on “Studies on Rabbit Reticulocytc Ribosomes”. Preceding paper is by Chatterjce, S.K., Kazemie, M., Matthaei, H., noppe-Seyler’s Z. Physiol. Chem.345, 481 (1973).     

Interaction of puromycin with acceptor site of human placenta 80 S ribosomes

The complex N-AcPhe-tRNA(Phe).poly(U).80 S ribosome from human placenta was treated with puromycin taken in various concentrations. Based on the kinetic data of N-acetylphenylalanyl-puromycin formation, the association constant of puromycin with the acceptor site of the ribosome was estimated to be (3.96 +/- 0.84) x 10(4) M-1 at 37 degrees C.     

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.     

Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine-Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

Covalent attachment of poly (U) template to 40S - mammalian ribosomal subunits

When 40S subunits are irradiated at 254nm in presence of [³H] poly (U), formation of a 40S subunit-poly (U) complex can be demonstrated either by filtration technique at low Mg⁺⁺ concentration or by polyacrylamide gel electrophoresis. No stable complex was detected using unirradiated samples under the same conditions. Electrophoresis of this complex in the presence of dodecyl sulfate showed that part of the poly (U) directly associates with 18S RNA. This association is not through proteins, since it is not disrupted by pronase treatment.     

Available sulphydryl groups of mammalian ribosomes in different functional states

The numbers of sulphydryl groups on NH₄Cl-washed rat liver polyribosomes in different functional states were measured under carefully standardized conditions with ¹⁴C-labelled N-ethylmaleimide and ³⁵S-labelled 5,5-dithio-bis(2-nitrobenzoic acid). Ribosomes denatured with urea had 120 titratable sulphydryl groups, 60 on each subunit, whereas native ribosomes invariably showed fewer available sulphydryl groups. Ribosomes stripped of transfer RNA (S-type ribosomes) had 55 available sulphydryl groups. Ribosomes bearing the growing peptidyl-tRNA at the acceptor site had 41 sulphydryl groups available. If these A-type ribosomes were labelled with ¹⁴C-labelled N-ethylmaleimide and dissociated into subunits, 23 of the labelled sulphydryl groups were found on the 60 S subunit and 19 on the 40 S subunit. After translocation of the peptidyl-tRNA to the donor position on ribosomes (D ribosomes), the number of available sulphydryl groups increased to 72, of which 43 were on the 60 S subunit and 29 on the 40 S subunit. This demonstrates that both subunits participate in the change of peptidyl-tRNA from the A to D positions. When the D ribosomes were reacted with EF2 (elongation factor) and GTP, the available sulphydryl groups increased to 82; addition of EF2 alone or with GDP, GDPCP or ATP failed to cause this increase, which has accordingly been attributed to an energy-dependent conformational change in the ribosome.Ribosomes were reconstructed from subunits with poly(U) and Phe-tRNA. In the presence of poly(U) only, a ribosome with 55 available SH groups was formed, thus corresponding to the stripped ribosomes. When both poly(U) and Phe-tRNA were present, a ribosome was formed with 44 available sulphydryl groups, corresponding approximately to an A-type ribosome. Since no EF1 or GTP was used in reconstructing this ribosome, these data indicate that the conformation of A-type ribosomes is not dependent on EF1 or GTP, but is due to the presence of tRNA at the acceptor site.We therefore incline to the view that the observed changes in available SH groups reflect conformational changes, with an opening up of ribosome structure as it progresses from having the peptidyl-tRNA at the A position to the D position and then binds EF2 and GTP, followed by a restoration of the more compact from when the incoming aminoacyl-tRNA is then bound.     

Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function.

The reaction pattern with N-[¹⁴C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg²⁺ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg²⁺-dependent. The reaction at 10 mm-Mg²⁺ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg²⁺; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg²⁺ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg²⁺. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg²⁺ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.     

The effect of tRNA binding on the structure of 5 S RNA in Escherichia coli. A chemical modification study

The structure of 5 S RNA within the 70 S ribosome from Escherichia coli was studied using the chemical reagent kethoxal (alpha-keto-beta-ethoxybutyraldehyde) to modify accessible guanosines. The modification pattern of 5 S RNA from free 70 S ribosomes was compared with that of poly(U) programmed ribosomes where tRNA had been bound to both the A- and P-sites. Binding to the ribosomal A-site was achieved enzymatically using the elongation factor Tu and GTP in the presence of deacylated tRNA which blocks the ribosomal P-site. Modified guanosines were identified after partial RNase T1 hydrolysis and separation of the hydrolysis products on sequencing gels. Binding of tRNA to the ribosome leads to a strong protection of 5 S RNA guanosine G-41 and to some degree G-44 from kethoxal modification. The limited RNase T1 hydrolysis pattern provides evidence for the existence of a 5 S RNA conformation different from the known 5 S RNA A- and B-forms which are characterized by their gel electrophoretic mobility. The importance of 5 S RNA for the binding of tRNA to the ribosome is discussed.     

A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA _inff ^supMet . Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA _inff ^supMet . Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU₃ as mRNA, elongator-tRNAs competed with fMet-fRNA _inff ^supMet and with tRNA _inff ^supMet . fMet-tRNA _inff ^supMet reacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU₃ complex at the A site only if fMet-tRNA _inff ^supMet or tRNA _inff ^supMet filled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.     

Binding of 30S Ribosome Induces Single-stranded Conformation Within and Downstream of the Expression Platform in a Translational Riboswitch

Translational riboswitches are bacterial gene regulatory elements found in the 5′-untranslated region of mRNAs. They operate through a conformational refolding reaction that is triggered by a concentration change of a modulating small molecular ligand. The translation initiation region (TIR) is either released from or incorporated into base pairing interactions through the conformational switch. Hence, initiation of translation is regulated by the accessibility of the Shine-Dalgarno sequence and start codon. Interaction with the 30S ribosome is indispensable for the structural switch between functional OFF and ON states. However, on a molecular level it is still not fully resolved how the ribosome is accommodated near or at the translation initiation region in the context of translational riboswitches. The standby model of translation initiation postulates a binding site where the mRNA enters the ribosome and where it resides until the initiation site becomes unstructured and accessible. We here investigated the adenine-sensing riboswitch from Vibrio vulnificus. By application of a ¹⁹F labelling strategy for NMR spectroscopy that utilizes ligation techniques to synthesize differentially ¹⁹F labelled riboswitch molecules we show that nucleotides directly downstream of the riboswitch domain are first involved in productive interaction with the 30S ribosomal subunit. Upon the concerted action of ligand and the ribosomal protein rS1 the TIR becomes available and subsequently the 30S ribosome can slide towards the TIR. It will be interesting to see whether this is a general feature in translational riboswitches or if riboswitches exist where this region is structured and represent yet another layer of regulation.