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Transferrin is a glycoprotein functioning in iron transport in higher eukaryotes, and consists of two highly homologous domains. To study the function of the glycan residues attached exclusively to the C-terminal domain, we have constructed a plasmid allowing production of nonglycosylated human transferrin in Escherichia coli. By molecular biological and genetic techniques, production was stepped up to 60 mg/l. Similar plasmids were constructed for production of the two half-transferrins. The recombinant proteins accumulate in inclusion-body-like aggregates, where they appear to bind iron without causing bacteriostasis. Proteins active in iron binding have been purified from these inclusion bodies.

To activate expression of a human transferrin (Tf)-encoding cDNA in Escherichia coli by translational coupling, it was placed in an expression plasmid downstream from a 5'-terminal fragment from the replicase (R)-encoding gene of bacteriophage MS2. The resulting construct was found to produce, besides the desired Tf, a protein with the mobility of a fusion product (RTf) of the N-terminal R fragment and Tf. Analysis of available mutants showed that this fusion results from +1 ribosomal frameshifting at the end of the R reading frame. This region contains the sequence, CCC.UGA, suggesting that before termination occurs, tRNA(Pro) may dislodge from the CCC codon and reassociate with the +1 triplet CCU. By further site-directed mutagenesis, we demonstrate that both the CCC codon and the termination codon are indeed required for the observed 2-4% frameshifting. When either triplet is changed, the frequency of frameshifting drops to 0.3% or less. These results classify CCC.UGA as a new '+1 shifty stop'.

U.G basepairs are well-established elements of RNA structure. The geometry of this pair is different, however, from classical Watson-Crick basepairs. This leads to an unusual stacking of the basepair: overlap with the basepair at the 5' side of the U (and the 3' side of the G) is strong (stacked) while it is weak with the basepair on the other side (destacked). The closure of an RNA helix by a U.G pair will be energetically unfavourable when the U residue occupies the 5' end. In transfer RNA there is a strong selection against a 'destacked' U.G pair at helix ends. In the 16S rRNA model of Escherichia coli there are 72 U.G pairs of which 36 or 22 occupy a helix end, depending on how such an end is defined. There is a slight preference for 'stacked' U.G's in these positions. It is remarkable, however, that of 13 very conserved U.G pairs in the 16S (-like) rRNA, 7 occur at helix ends and that 5 of these have the 'destacked' configuration. It is suggested that these pairs, if they exist at all in a hydrogen-bounded form, are stabilized by co-axial stacking with other helices or by interaction with a protein.

In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity. The amount varied according to the assay and mutant, but was never less than 30% and was generally above 50%. Therefore, neither the conserved A1518 nor A1519 residues are essential for ribosome function. The mutant ribosomes could also be methylated by the ksgA methyltransferase to 70-120% of the expected amount. Thus, neither of the A residues is required for methylation of the other, ruling out any obligate order of methylation of A1518 and A1519.

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.

Fragments comprising the 49 nucleotides from the 3'-end have been purified from 16S ribosomal RNA of wild-type Escherichia coli and from a kasugamycin-resistant mutant that specifically lacks dimethylation of two adjacent adenines near the 3'-terminus. These fragments, obtained after treatment of ribosomes in vitro with the bacteriocin cloacin DF13, were used to study the effect of the methyl groups on the temperature dependent unfolding of double-stranded regions. Both fragments contain at least 3 independent melting transitions, of which the one with the highest Tm corresponds with the unfolding of a nine-basepair long central hairpin. Dimethylation of the adenines in the loop of this hairpin lowers the melting temperature (Tm) by approximately 2 degrees C at 0.2 M NaCl and by about 5 degrees C at 0.15 M NaCl. It is suggested that m6(2)Am6(2)A is more antagonistic to loop formation that ApA and that the function of the methyl groups is to help to destabilize the 3'-terminal hairpin in 16S rRNA in order to facilitate intermolecular interactions.

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with our previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A. 73, 702--706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can be compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subunit interaction.

We have replaced the ribosomal binding site (RBS) of the lacZ gene of E. coli by those of the maturation (A) gene of phage MS2 and that of the tufA gene. Both RBSs contain a GUG initiation codon. The expression with the tufA RBS is at least 25-fold higher than with the phage RBS. Changing the GUG into AUG results in a 3-fold increase in expression in both cases. In general, higher expression is accompanied by an increase of lac-specific mRNA. It is argued that this is a consequence of the more efficient translation of the mRNA.

30S ribosomes were isolated from a kasugamycin resistant mutant of E. coli that lacks methylgroups on two adjacent adenines in 16S ribosomal RNA. These ribosomes were methylated in vitro with a purified methylating enzyme and 5-S-adenosyl-(13C-methyl)-L-methionine chloride ((13C-methyl)-SAM) as methyldonor. After in situ cleavage of the 16S ribosomal RNA by the bacteriocin cloacin DF13, the 49 nucleotide fragment from the 3'-end of the RNA was isolated. The carbon-13 nuclear magnetic resonance spectra of the fragment at various temperatures were compared with those of 6-N-dimethyladenosine (m6(2)A) and 6-N-dimethyladenylyl-(3' leads to 5')-6-N-dimethyladenosine (m6(2)Am6(2)A). The data show that the two methylated adenines, which are part of a four membered hairpin loop, show a strong tendency to be stacked in analogy to the dinucleotide m6(2)Am6(2).

The effect of the presence or absence of methyl groups on the N6 atoms of two adjacent adenosines near the 3' end of 16 S rTNA of Escherichia coli on initiation of protein biosynthesis has been studied using wild type (methylated) and kasugamycin-resistant (unmethylated) E. coli ribosomes (see preceding paper (Poldermans, B., Goosen, N., and Van Knippenberg, P. H. (1979) J. Biol. Chem. 254, 9085--9089)). Conditions of pH, temperature, and ionic strength at which binding of fMet-tRNA to ribosomes proceeds maximally are the same for wild type and mutant ribosomes. Mg2+- and factor-dependent dissociation of ribosomes as well as the association of the subunits is also the same for methylated and unmethylated ribosomes. Binding of fMet-tRNA to wild type and to mutant 70 S ribosomes requires the same amount of the three initiation factors. However, optimal fMet-tRNA binding to unmethylated 30 S ribosomes needs more of initiation factor 3 than does binding to methylated 30 S ribosomes, provided that initiation factor 1 is absent. This difference is completely abolished when mutant 30 S ribosomes are methylated using purified methylase from the wild type strain and the methyl donor S-adenosylmethionine.

Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF-2-dependent binding of fMET-tRNA to 30-S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230-234]. The N-formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template. This paper shows that N-acetylated Phe-tRNAPhe (AcPhe-tRNA), but not Phe-tRNAPhe or tRNAPhe, competes with fMET-tRNA for binding to poly(U)-programmed 30-S ribosomal subunits. The two species of N-blocked aminoacyl-tRNA are bound to poly(U)-programmed and poly(X)-programmed 30-S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMET-tRNA or AcPhe-tRNA, whereas with poly(X) there is a 2-3-fold preference for fMET-tRNA. The initiation factor IF-2, which is strictly required for the binding of N-blocked aminoacyl-tRNAs, has a higher affinity for fMET-tRNA than for AcPhe-tRNA. It is concluded that (a) interaction of the 30-S ribosomal subunit with poly(U) or poly(X) leads to IF-2-dependent binding of N-blocked aminoacyl-tRNA; (b) the initiation factor IF-2-discriminates in favour of fMET-TRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N-blocked aminoacyl-tRNA.

Fragments of 16S ribosomal RNA from E. coli and B. stearothermophilus, respectively comprising the 49 and 52 3' terminal nucleotides have been studied thermodynamically using high sensitivity differential scanning calorimetry. The fragments were isolated after cleavage of 16S rRNA in the ribosome by the bacteriocin cloacin DF13. Comparison of the thermal properties of the E. coli fragments with those derived from a kasugamycin resistant mutant, which specifically lacks dimethylation of two adjacent adenosines was employed to study the effect of the methylgroups on the thermal stability. Both E. coli species exhibit similar complex melting patterns with several transitions. Overall molar transition enthalpies are equal and do not depend significantly on buffer conditions (120 kcal/mol at 15 mM Na+ to 136 kcal/mol at 215 mM Na+). However, the transition with the highest Tm, corresponding to unfolding of a nine basepair central helix is lowered by the dimethylation of the adenines in the four-membered loop. This decrease amounts to 4 degrees C at 15 mM Na+ and 2 degrees C at 215 mM Na+. The corresponding nine basepair long hairpin in the Bacillus fragment melts at a temperature of 70 degrees C at 15 mM Na+. This Tmax is much higher than expected on the basis of the sequence in the hairpin.

Bacterial, cytoplasmic and organellar ribosomes from a wide phylogenetic spectrum of organisms have a characteristic m6(2)Am6(2)A structure near the 3' end of the RNA of the small ribosomal subunit (SSU). We have studied one of the few exceptions to this extremely conserved post-transcriptionally modified sequence, i.e. dimethylation of only one of the two A's in chloroplasts from Euglena gracilis. It was established that only the A closest to the 5' end is dimethylated, the other one being unmodified. The methylation reaction was studied in vitro using ribosomes from a kasugamycin resistant mutant (ksgA) of Escherichia coli and purified methyl-transferase. Using limited amounts of the methyl donor S-adenosylmethionine (SAM) a partial level of methylation (50% of control) was attained. It is shown that in this case the 3' proximal A is dimethylated while the other is not. This suggests that dimethylation takes place in two successive stages. Apparently in E. gracilis chloroplasts the first stage of methylation does not occur.

Binding of the polynucleotides poly(U), poly(X) and poly(dT) to 30 S ribosomes of Escherichia coli triggers IF2-dependent binding of initiator-tRNA (fMet-tRNA) to these particles. Poly(A) and poly(C) are inactive. A minimum chain-length of approximately 100 residues in poly(U) is required for full activity in fMet-tRNA binding, although much shorter polymers bind tightly to 30 S particles and do stimulate the binding of acPhe-tRNA. The stimulation of fMet-tRNA binding to 30 S ribosomes is strongly reduced under conditions where the polynucleotides adopt secondary structure. Complexes containing fMet-tRNA and the non-cognate codon UUU or XXX are destabilized by IF3, whereas the formation of such a complex containing an AUG codon is slightly enhanced by the factor. Consistent with previous observations, it was found that all model initiation complexes containing acPhe-tRNA are strongly destabilized by IF3, even when the cognate codon (UUU) is present. Our results suggest that IF3 counteracts 'unnatural' initiation events in vitro and suggest a regulatory role for this factor in vivo.

The RNA of small ribosomal subunits contains a conserved stem-loop structure near the 3' end. Characteristics for the hairpins are: (a) a nine-basepairs stem: (b) a conserved A-UU-G junction in the stem: (c) a conserved sequence Gm6(2)AM6(2)A sequence in the loop (except yeast mitochondria and mutants from bacteria). We are using UV-optics, micro-calorimetry and 500 MHz-NMR to investigate fragments of about 50 nucleotides cleaved from the 3' ends of small ribosomal subunit RNA's by bacteriocins. Our preliminary conclusions are: (1) Dimethylation of the adenines in the loop destabilizes the hairpin because of an increased stacking; (2) melting of the hairpin starts at the ends as well as in the middle at the A-UU-G junction; (3) basepair substitutions have an unexpectedly large effect on thermal stability.

The aminoglycoside kasugamycin, which has previously been shown to inhibit initiation of protein biosynthesis in vitro, also affects translational accuracy in vitro. This is deduced from the observation that the drug decreases the incorporation of histidine relative to alanine into the coat protein of phage MS2, the gene of which is devoid of histidine codons. The read-through of the MS2 coat cistron, due to frameshifts in vitro, is also suppressed by the antibiotic. In contrast, streptomycin enhances histidine incorporation and read-through in this system. The effects of kasugamycin take place at concentrations that do not inhibit coat protein biosynthesis. Kasugamycin-resistant mutants (ksgA) lacking dimethylation of two adjacent adenosines in 16 S ribosomal RNA, show an increased leakiness of nonsense and frameshift mutants (in the absence of antibiotic). They are therefore phenotypically similar to previously described ribosomal ambiguity mutants (ram).

Several mutants of B. stearothermophilus have been isolated that are resistant to the antibiotic kasugamycin. One of these is shown to lack dimethylation of two adjacent adenosines in the 16S ribosomal RNA. All mutants that were analyzed biochemically lack the enzyme that is able to methylate this site. Ribosomal sensitivity and resistance to kasugamycin in B. stearothermophilus is therefore, like in E. coli, closely connected with dimethylation of the adenosines.

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of 24 systematically modified analogues of adenosine 3',5'-monophosphate (cAMP) to enhance the synthesis of beta-galactosidase in glucose-repressed Escherichia coli strains KNBL 1001 and cpd- Crookes has been investigated. The properties of the analogues in comparison with cAMP are, with only two exceptions, alike in both strains. Two analogues, 7-deazaadenosine 3',5'-monophosphate (i.e. tubercidin 3',5'-monophosphate) and (Rp)-adenosine 3',5'-monothionophosphate, exhibit higher biological activity than cAMP. The latter analogue is 50-fold more active in both strains. Three analogues showed activities comparable to cAMP, four analogues were less active and 12 analogues were unable to antagonize catabolite repression. Structure-activity correlations showed that the 2'OH-, 3'O-, 5'O-, the negative charge and the 6-amino group cannot be modified without losing biological activity in vivo, while the N-1 and N-7 in adenine are not essential. The interaction with the catabolite gene activator protein is stereoselective for an unmodified axial exocyclic oxygen. The results are compared to those obtained with cAMP analogues in E. coli in vitro and those obtained with the same analogues in protein-kinase systems and Dictyostelium species. The model of McKay et al. [McKay, D.B., Weber, J.T. and Steitz, T.A. (1982) J. Biol. Chem. 257, 9518-9524] proposed for distinct chemical interactions of cAMP with the catabolite gene activator protein is discussed and supplemented by additional hydrogen bond interactions.

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.

Extensive complementarity is found between the 5' end of 16S ribosomal RNA and protein synthesis initiation sites of bacteriophage RNA. Hybrids can be constructed from base sequences of 16S-RNA and two initiation regions on phage RNA. A model is proposed for the involvement of 16S-RNA in the unfolding of hairpin loops containing the initiation codon AUG.

The electrophoretic mobility of RNA fragments derived from the 3'-end of 16S rRNA on slabs of polyacrylamide gel in the presence of urea is strongly influenced by dimethylation of the N6-aminogroup of two adjacent adenosines. This is not due to the presence of the methylgroups per se, but must be ascribed to an effect of methylation on long range intramolecular interactions at these denaturing conditions. When it is assumed that the electrophoretic mobilities of the RNA fragments in the polyacrylamide matrix are determined by the conformational state(s) of the fragments, dimethylation of the adenosines leads in the smaller fragments to a less compact average conformation and in the larger fragments to a more compact average conformation. An effort is made to comprehend the effects of adenosine dimethylation in terms of secondary structure based on nucleotide sequence.

The sequence and the position of post-transcriptionally modified residues of the 3' -terminal end of Bacillus stearothermophilus 16S ribosomal RNA have been determined from the fragment that is cleaved off by bacteriocin treatment. The fragment contains 52 nucleotides, as compared to the 49 nucleotides of the corresponding fragment from E. coli ribosomes, The additional nucleotides are present in the sequence UCU very next to the 3' -terminus as was published earlier (1). The remainder of the sequence is identical to the one of E. coli except at six positions, due to the UV melting properties of the colicin fragment from B. stearothermophilus in comparison to the same fragment of E. coli show that the RNA from the thermophile has a more stable secondary structure.

The strongest conserved part of the RNA of small ribosomal subunits is probably located near the 3' end. This paper reviews the primary and secondary structures of some 40 sequenced 3' termini and tries to classify these structures according to common features and differences. The regions under consideration contain at the 5' side an almost universal, supposedly single-stranded stretch of nucleotides with the sequence--AAGUCGUAACAAGGU--. This is followed by a stem-loop structure. The stem always contains 9 basepairs (including U-G pairs) and no mismatches or bulged nucleotides. The loop of the hairpin is either (m2)GGm62Am62A (bacteria, chloroplasts and mitochondria) or UGm62Am62A (cytoplasm). The hairpin is, in most cases, followed at the 3' side by--GGAUCA--. Next to it bacteria and chloroplasts contain the so-called "Shine and Dalgarno" sequence --CCUCC--. The stem region of the hairpin contains a conserved A-U U-G junction. The two basepairs between this junction and the loop are either of type 1 (G-C G-C) or type 2 (C-G C-G). Classification according to type links certain bacteria with mitochondria of yeast and plants and others with chloroplasts and with animal mitochondria.

Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 from Escherichia coli on the synthesis of the coat protein of the RNA-containing phages Qbeta and MS2, on that of an "early" and a "late" enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicate that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.

Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.

The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The previously reported requirement of ribosomal protein S1 for translation of phage RNA is now shown to be related to the involvement of the protein in initiation complex formation. The structure of the messenger RNA appears to be uniquely related to S1 function, since translation and initiation and midly unfolded phage RNA (by modification with formaldehyde) are independent of S1. It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA. A model is suggested for S1 function in both initiation of protein synthesis and initiation of phage RNA replication.

An inducible erythromycin resistance gene (erm) of Streptococcus pyogenes was introduced into Escherichia coli by transformation with a plasmid. The recipient E. coli cells were either kasugamycin sensitive (wildtype) or kasugamycin resistant (ksgA). The MIC values of erythromycin increased from 150 micrograms/ml to greater than 3000 micrograms/ml for E. coli. An extract of transformed cells, particularly a high-salt ribosomal wash, contained an enzyme that was able to methylate 23S rRNA from untransformed cells in vitro; however, 23S rRNA from transformed cells was not a substrate for methylation by such an extract. 165 rRNA and 30S ribosomal subunits of either the wild type or a kasugamycin resistant (ksgA) mutant were not methylated in vitro. Transformation of E. coli by the erm-containing plasmid led to a reduction of the MIC values for kasugamycin. This happened in wild-type as well as in ksgA cells. However, in vitro experiments with purified ksgA encoded methylase demonstrated that also in erm transformed E. coli, the ksgA encoded enzyme was active in wild-type, but not in ksgA cells. It was also shown by in vitro experiments that ribosomes from erm ksgA cells have become sensitive to kasugamycin. Our experiments show that in vivo methylation of 23S rRNA, presumably of the adenosine at position 2058, leads to enhanced resistance to erythromycin and to reduced resistance to kasugamycin. This, together with previous data, argues for a close proximity of the two sites on the ribosome that are substrates for adenosine dimethylation.

The 3' terminus of 16S rRNA has been implicated in the recognition of mRNA's by the ribosome. A fragment containing the 3'-terminal 49 nucleotides cleaved from the rRNA by cloacin DF13 was isolated in a pure form. The secondary structure of this fragment has been studied by measuring the high-resolution proton magnetic resonance spectra. The resonances observed at low field can be assigned to hydrogen-bonded iminoprotons of base-pairs present in the fragment. From the data we conclude that the rRNA fragment, under the conditions used, exists as a hairpin consisting of eight intramolecular base-pairs, the 3'-terminal dodecanucleotide being unpaired. The implications of these findings with respect to the function of the ribosomal protein S1 are discussed.

A vector (pKL203) was constructed which contains the promoter-operator region of the lacZ gene and the major part of the coding sequence of the lac operon. The lacZ translation initiation signals [Shine-Dalgarno (SD) sequence and AUG codon] were deleted, and in their place a synthetic linker sequence was inserted, providing single restriction sites for SmaI and BamHI. With this vector constructions were made in which initiation signals of other prokaryotic genes (phage MS2 maturation protein, phage Q beta A2 gene and tufB gene) were fused to the lacZ gene, giving rise to various fusion proteins. The introduction of N-terminal amino acids (aa) in beta-galactosidase (beta-gal) which differ from the wild-type aa invariably leads to an enzyme with a strongly reduced thermostability as compared to the wild-type enzyme. Therefore an immunoprecipitation method was used to measure the amount of fusion protein. It was found that these amounts varied strongly from one construction to another. Concomitant determinations of the amounts of lac-operon-specific mRNA showed an unexpectedly large variation among the clones. No strict correlation could be found between the level of lac mRNA and beta-gal production. Per molecule of lac mRNA, translation appears to be most efficient when the homologous lacZ initiation signal is present.

The 49 nucleotides fragment derived from the 3' end of 16S rRNA by cloacin DF13, is not cleaved by ribonuclease T1 at a guanosine residue tha is present at 27 nucleotides from the 3' terminus (position 115 in 16S rRNA). Analysis of the isolated nucleotide indicates that it is a modified G residue. In vivo labeling with (3H)methionine shows that this G is methylated and co-chromatography with markers reveals that it is N2-methylguanosine.

Specific in situ cleavage of 16S rRNA of E. coli has been accomplished by in vitro treatment of 70S ribosomes ("tight couples") with the bacteriocin cloacin DF13. The defective ribosomes, which have fully lost their ability to sustain polypeptide synthesis, are still able to form initiation on complexes with MS2 RNA, but the kinetics are altered. This is apparently due to an improper functioning of initiation factor IF-1, for the defective ribosomal couples respond normally to dissociation by IF-3 but the dissociation is not stimulated by IF-1. The initiation complexes formed with defective ribosomes are fully reactive with puromycin. Their ability to bind alanyl-tRNA is reduced by about 50% at all concentrations of elongation factor Tu studied. Cleavage of the 16S rRNA, not the release of the terminal fragment from the ribosome, causes the block of protein synthesis and the aberrations observed during initiation and elongation.

The conformation of the colicin fragment of E. coli 16S rRNA was probed with various nucleases and with the adenosine-specific reagent diethylpyrocarbonate (DEP). The results confirm the presence of a stable central hairpin in the colicin fragment and a weaker additional secondary structure involving the regions 5' and 3' to this hairpin. By monitoring DEP accessibility at various stages of heat-denaturation sequential unfolding of individual base pairs was followed. In agreement with previous results it could be shown that dimethylation of the two adjacent adenosines in the hairpin loop (a feature in virtually all ribosomes) leads to a destabilization of the hairpin helix. Accessibilities of G residues, involved in the weaker additional secondary structure is anomalous. One G residue is sensitive to the single strand specific RNase T1 and insensitive to DEP, while the situation is reversed for the adjoining G residue. The strong reaction of the latter G-residue with DEP is unusual and indicates a very special conformation.

The ksgA gene of Escherichia coli encodes a methyltransferase (MeT) that specifically dimethylates two adjacent adenosines near the 3' end of 16S RNA in the 30S particle. Its inactivation leads to kasugamycin (Ksg) resistance. Several plasmids were constructed with inserts which complemented chromosomal ksgA mutations. One of these inserts was sequenced and found to contain an open reading frame (ORF) sufficient to code for the previously identified 30-kDal MeT. We have compared the amino acid (aa) sequence of the ksgA-encoded enzyme with three published sequences of MeT involved in dimethylation of an adenosine residue in 23S RNA and rendering the organisms resistant to the MLS antibiotics. The homologous patches in the sequences of all four enzymes suggest that those might correspond to contact points for the common substrates, e.g., for the adenosine residue(s) and S-adenosylmethionine (SAM).

Various plasmids that contain the Escherichia coli ksgA gene, which encodes a 16S rRNA adenosine dimethyltransferase (methylase), were constructed. In one of these plasmids, the DNA encoding the N-terminal part of the methylase was fused to the lacZ gene, and in another construct, the ksgA gene contained a deletion which resulted in a truncated version of the methylase. When a cell contained one plasmid directing the synthesis of the intact, active methylase and another plasmid encoding the methylase-beta-galactosidase protein, production of the latter product became strongly reduced. Likewise, synthesis of the truncated version of the methylase was diminished when the cell at the same time contained a plasmid producing the complete enzyme. These results were partly substantiated by in vitro experiments with a coupled transcription-translation assay system. By using a recently developed gel electrophoresis system for measuring protein-nucleic acid interactions, a specific binding of the ksgA methylase with its own mRNA could be established. Our results demonstrate that the expression of the ksgA gene can be, at least partly, autogenously controlled at the level of translation.