Location of protein S1 of Escherichia coli ribosomes at the ''A''-site of the codon binding site. Affinity labeling studies with a 3''-modified A-U-G analog.

An affinity analog with a 5-bromoacetamido uridine 5'-phosphate moiety bonded to the 3' end of A-U-G has been prepared with the aid of polynucleotide phosphorylase. This 3'-modified, chemically reactive A-U-G analog was used to probe the ribosomal codon binding site. The yield of the reaction depended strongly on the ribosomal source and was sensitive to salt-washing ribosomes. The major crosslinking product was identified to be protein S1. Since the reaction of this 3'-modified A-U-G programmed ribosomes for Met-tRNA-Met-M binding, it is concluded that protein S1 is located at or near the 3'-side of the ribosomal codon binding site.     

Identification of cysteine-10 of protein S18 as part of the mRNA-binding site of Escherichia coli ribosomes by affinity-labeling studies with a chemically reactive A-U-G analog.

The reaction of a bromoacetamidophenyl derivative of the initiation codon A-U-G (A-U-G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A-U-G-directed fMet-tRNAfMet binding into the puromycin-sensitive site of ribosomes and stimulates elongation-factor-dependent binding of Met-tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl-tRNA binding site of E. coli ribosomes. Peptide as well as amino acid analysis shows that the reaction between A-U-G and ribosomes took place at cysteine-10 of protein S18. A-U-G could not be crosslinked to ribosomal proteins of the temperature-sensitive E. coli strain 258ts, where arginine-11 of protein S18 is replaced by a cysteine residue.     

Crosslinking of chemically reactive A-U-G analogs to ribosomal proteins within initiation complexes.

A-U-G analogs, either reactive on their 5′ or their 3′ side, were employed in affinity labeling of the ribosomal A-U-G binding site. These experiments have been carried out such that the chemically reactive A-U-G analog became covalently bonded to ribosomal proteins only in the presence of fMet-tRNA_f^Met and initiation factors. Subsequent radioimmunodiffusion of A-U-G-labeled proteins identified proteins IF3, S1, S18, S21 and L11 as being in the neighborhood of the ribosomal codon binding site. A location of reactive sites of these proteins relative to the P or A site bound codon is, however, not clear.The A-U-G labeling results are quantitatively as well as qualitatively very different in the absence or presence of fMet-tRNA_f^Met. It is concluded, therefore, that fMet-tRNA_f^Met directs A-U-G into its final binding site. Streptomycin cannot release fMet-tRNA_f^Met from initiation complexes which contain irreversibly bound 5′- {4-(bromoacetamido)phenylphospho}-adenylyl-(3′–5′)-uridylyl-(3′–5′)-guanosine. This suggests that codon-anticodon interaction between A-U-G and fMet-tRNA_f^Met is still intact in the P site of the ribosome.     

Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation

The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.     

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

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.     

Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.     

Basepairing of oligonucleotides to the 3'' end of 16S ribosomal RNA is not stabilized by ribosomal proteins

We have studied the binding of the octanucleotide (5'-3')d(AAGGAGGT) which is fully complementary to the 3' end of 16S ribosomal RNA, to ribosomes and to the isolated target sequence (5'-3') (ACCUCCUUA). The binding constant for 30S or 70S ribosomes is (5 +/- 2) X 10(7) mol-1, whereas the duplex containing the octa- and the nonanucleotide has an association constant of (6 +/- 3) X 10(7) mol-1. The two values are the same within the experimental error. This result suggests that basepairing at the 3' end of 16S rRNA is not stabilized by ribosomal proteins.     

Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.     

Factor requirements for translation initiation on the Simian picornavirus internal ribosomal entry site

The Simian picornavirus type 9 (SPV9) 5'-untranslated region (5' UTR) has been predicted to contain an internal ribosomal entry site (IRES) with structural elements that resemble domains of hepacivirus/pestivirus (HP) IRESs. In vitro reconstitution of initiation confirmed that this 5' UTR contains an IRES and revealed that it has both functional similarities and differences compared to HP IRESs. Like HP IRESs, the SPV9 IRES bound directly to 40S subunits and eukaryotic initiation factor (eIF) 3, depended on the conserved domain IIId for ribosomal binding and consequently for function, and additionally required eIF2/initiator tRNA to yield 48S complexes that formed elongation-competent 80S ribosomes in the presence of eIF5, eIF5B, and 60S subunits. Toeprinting analysis revealed that eIF1A stabilized 48S complexes, whereas eIF1 induced conformational changes in the 40S subunit, likely corresponding to partial opening of the entry latch of the mRNA-binding channel, that were exacerbated by eIF3 and suppressed by eIF1A. The SPV9 IRES differed from HP IRESs in that its function was enhanced by eIF4A/eIF4F when the IRES was adjacent to the wild-type coding sequence, but was less affected by these factors or by a dominant negative eIF4A mutant when potentially less structured coding sequences were present. Exceptionally, this IRES promoted binding of initiator tRNA to the initiation codon in the P site of 40S subunits independently of eIF2. Although these 40S/IRES/tRNA complexes could not form active 80S ribosomes, this constitutes a second difference between the SPV9 and HP IRESs. eIF1 destabilized the eIF2-independent ribosomal binding of initiator tRNA.     

Evidence that the requirements for ATP and wheat germ initiation factors 4A and 4F are affected by a region of satellite tobacco necrosis virus RNA that is 3' to the ribosomal binding site

A cDNA containing the complete genome of satellite tobacco necrosis virus (STNV) RNA was constructed and cloned into a plasmid vector containing the T7 polymerase promotor. A second clone containing the first 54 nucleotides from the 5' end, which includes the ribosome binding site, was also constructed. RNAs were transcribed from these plasmids (pSTNV1239 and pSTNV54) and tested for their ability to bind to wheat germ 40 S ribosomal subunits in the presence of wheat germ initiation factors eIF-4A, eIF-4F, eIF-4G, eIF-3, eIF-2, Met-tRNA, ATP, and guanosine 5'-(beta, gamma-imino)triphosphate (GMP-PNP). Maximal binding of the STNV RNA transcribed from pSTNV1239 is obtained only in the presence of all the initiation factors and ATP. In contrast, close to maximal binding of STNV RNA transcribed from pSTNV54 is obtained in the absence of eIF-4A, eIF-4F, eIF-4G, and ATP. A series of deletion clones from the 3' end of the STNV cDNA was prepared, and the requirements for binding to 40 S ribosomal subunits were determined. STNV RNAs containing more than 134 nucleotides from the 5' end require eIF-4A, eIF-4F, eIF-4G, and ATP for maximal binding to 40 S ribosomal subunits, whereas STNV RNAs containing 86 nucleotides or less no longer require ATP and these factors. These findings indicate that a region 3' to the initiation codon affects the requirements for eIF-4A, eIF-4F, eIF-4G, and ATP.     

Ribosome binding to a 5' translational enhancer is altered in the presence of the 3' untranslated region in cap-independent translation of turnip crinkle virus

Plus-strand RNA viruses without 5' caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5' UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5' UTR sequences. Addition of 80S ribosomes to the 5' UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5' end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5'-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5' and 3' sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5' and 3' UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5' UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5' and 3' UTRs of TCV.     

Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.     

Inhibition of chloramphenicol binding to Escherichia coli 70S ribosomes by 2'(3')-O-aminoacyl-dinucleoside phosphates derived from the aminoacyl-tRNA acceptor terminus.

The effect of 2' and 3'-O-aminoacyl-dinucleoside phosphates cytidylyl(3'-5')-2'(3')-O-L-phenyl-alanyladenosine (I), cytidylyl(3'-5')-3'-deoxy-2'-O-L-phenylalanyladenosine (IIa), cytidylyl(3'-5')-2'-deoxy-3'-O-L-phenylalanyladenosine (IIIa), cytidylyl(3'-5')-3'-deoxy-2'-O-glycyladenosine (IIb), cytidylyl(3'-5')-2'-deoxy-3'-O-glycyladenosine (IIIb), cytidylyl(3'-5')-3'-deoxy-2'-O-L-leucyladenosine (IIc), cytidylyl(3'-5')-2'-deoxy-3'-O-L-leucyladenosine (IIIc), cytidylyl(3'-5')-3'-O-L-phenylalanyladenosine (IIId) as analogs of the 2'(3')-aminoacyl-tRNA termini, on chloramphenicol binding to 70S Excherichia coli ribosomes was investigated. The association constants (Kb) of the investigated compounds were determined by the equilibrium dialysis method. Based on the constancy of Kb over the range of inhibitor concentration, it was determined that the binding site of the 2' isomers IIa-IIc overlaps with the chloramphenicol site, whereas the variability of Kb for the 3' isomers IIIb, IIIc and especially IIIa seems to indicate that they do not achieve a complete fit. The consistently higher values of the Kb values for the 3' isomers IIIa-IIIc relative to that of the 2' isomers IIa-IIc also indicate a stabilization of the binding of the former due to a specific interaction between its amino acid portion and a ribosomal site.     

Affinity labeling of the P site of Drosophila ribosomes: a comparison of results from (bromoacetyl)phenylalanyl-tRNA and mercurated fragment affinity reactions

The binding site of the peptidyl group of peptidyl-tRNA in the P site of Drosophila ribosomes was probed with (bromoacetyl)phenylalanyl-tRNA (BrAcPhe-tRNA). This affinity label binds specifically to the P site by virtue of its ability to participate in peptide bond formation with puromycin following its attachment to ribosomes. As many as nine ribosomal proteins may be labeled under these conditions; however, the majority of the labeling is associated with three large-subunit proteins and two small-subunit proteins. Two of the large-subunit proteins, L4 and L27, are electrophoretically very similar to the proteins labeled by the same reagent in Escherichia coli ribosomes L2 and L27. Reexamination by a different two-dimensional gel system of the ribosomal components labeled by a second P site reagent, the 3' pentanucleotide fragment of N-acetylleucyl-tRNA which is derivatized to contain mercury atoms at the C-5 position of all three cytosine residues, shows two major and three minor labeled proteins. These proteins, L10/L11, L26, S1/S4, S13, and S20, are likely present in the binding site of the 3' end of peptidyl-tRNA, a site that appears to span both subunits. These results have allowed us to construct a model for the protein positions in and near the peptidyl-tRNA binding site of Drosophila ribosomes.     

Crosslinking of mRNA Analog,pUUAGUAUUUAUU Derivative with Photoactivatable Group at the Guanosine Residue,with Human 80S Ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg²⁺, or 4 mM Mg²⁺ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840–1849 of 18S rRNA, but in the complexes formed with participation of Phe-tRNA^Phe (where the G residue carrying the arylazido group occupied position –3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position –3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg²⁺ concentration and the presence of polyamines.     

Blocking of the initiation of protein biosynthesis by a pentanucleotide complementary to the 3' end of Escherichia coli 16 S rRNA.

Hydrogen bonding between the 3' terminus of 16 S rRNA (... C-A-C-C-U-C-C-U-U-A-OH3) and complementary sequences within the initiator region of mRNA may be a crucial event in the specific initiation of protein biosynthesis (Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1342-1346; Steitz, J. A., and Jakes, K. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4734-4738). Using equilibrium dialysis, we have studied the binding of G-A-dG-dG-U (which is complementary to the 3' end of 16 S rRNA and which has been synthesized enzymatically) to initiation factor-free Escherichia coli ribosomes. We have also investigated the effects of the pentanucleotide on initiation reactions in E. coli ribosomes. G-A-dG-dG-U has a specific binding site on the 30 S ribosome with an association constant of 2 x 10(6) M-1 at 0 degrees C. G-A-dG-dG-U inhibits the R17 mRNA-dependent binding of fMet-tRNA by about 70%, both with 70 S ribosomes and 30 S subunits. In contrast, the A-U-G-dependent initiation reaction and the poly(U)-dependent Phe-tRNA binding was not affected by the pentanucleotide with both ribosomal species.     

Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence

It has previously been proposed that Escherichia coli ribosomal protein S1 is required for the translation of highly structured mRNAs. In this study, we have examined the influence of structural features at or near the start codon of different mRNAs. The requirement for ribosomal protein S1 for translation initiation was determined when (i) the ribosome-binding site (RBS) was either preceded by a 5' non-translated leader sequence; (ii) the RBS was located 5' proximal to a mRNA start codon; and (iii) the start codon was the 5' terminal codon as exemplified by leaderless mRNAs. In vitro translation studies revealed that the leaderless lambda cl mRNA is translated with Bacillus stearothermophilusribosomes, naturally lacking a ribosomal protein S1 homologue, whereas ompA mRNA containing a 5' leader is not. These studies have been verified by toeprinting with E. coli ribosomes depleted for S1. We have shown that S1 is required for ternary complex formation on ompA mRNA but not for leaderless mRNAs or for mRNAs in which the RBS is close to the 5' end.     

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.     

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.     

Localization of the elongation factor Tu binding site on Escherichia coli ribosomes

Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.