P.H. Van Knippenberg

Function of Escherichia coli ribosomal protein S1 in translation of natural and synthetic messenger RNA

Abstract The effect of Escherichia coli ribosomal protein S1 on translation has been studied in S1-depleted systems programmed with poly(U), poly(A) and MS2 RNA ‡ . The translation of the phage RNA depends strictly on the presence of S1. Optimum poly(U)-directed polyphenylalanine synthesis and poly(A)-programmed polylysine synthesis also require S1. Excess S1 relative to ribosomes and messenger RNA results in inhibition of translation of MS2 RNA and poly(U), but not of poly (A). In the case of phage RNA translation, this inhibition can be counteracted by increasing the amount of messenger RNA. Three other 30 S ribosomal proteins (S3, S14 and S21) are also shown to inhibit MS2 RNA translation. The effects of S1 on poly(U) translation were studied in detail and shown to be very complex. The concentration of Mg 2+ in the assay mixtures and the ratio of S1 relative to ribosomes and poly(U) are crucial factors determining the response of this translational system towards the addition of S1. The results of this study are discussed in relation to recent developments concerning the function of this protein.

Functional heterogeneity of the 30 s ribosomal subunit of Escherichia coli: III. Requirement of protein s1 for translation

Abstract Poly(U)-dependent polyphenylalanine synthesis is completely dependent on the presence of ribosomal protein S1. Polysomes generated under the direction of poly(U) contain approximately one molecule of S1 per ribosome. Isolation of 30 S ribosomes from poly(U)-generated polysomes by a procedure requiring a low concentration of Mg 2+ (0·25 mM) results in loss of S1. S1 is probably also required for the phage RNA-dependent binding of formylmethionyl-tRNA. The data are discussed in relation to current concepts of the functional aspects of ribosome heterogeneity.

Increased translational fidelity caused by the antibiotic kasugamycin and ribosomal ambiguity in mutants harbouring the ksgA gene.

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).

Functional heterogeneity of the 30S ribosomal subunit ofEscherichia coli

SummaryNative 30S ribosomal subunits fromEscherichia coli are deficient in fractional protein S21, which is present on the monosome and polysome-derived 30S subunits. The presence of S21 prevents the binding of Fmet-tRNA if and only if 50S subunits are present. In contrast, proteins S2, S3 and S14 stimulate the binding of Fmet-tRNA. These results have been used to rationalize other data concerning the mechanism of Fmet-tRNA binding by ribosomes. In addition, the present data indicate that the 30S ribosomal subunits are heterogeneousin vivo as well asin vitro.

Kasugamycin resistant mutants of Bacillus stearothermophilus lacking the enzyme for the methylation of two adjacent adenosines in 16S ribosomal RNA

SummarySeveral 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.

An initiation factor causing dissociation of E. coli ribosomes

Purification of crude initiation factors, essential for polypeptide synthesis in cell‐free systems of E. coli, yielded a fraction DF which causes dissociation of 70 S ribosomes. Its stoichiometric action on 70 S ribosomes is antagonized by increasing Mg2+ concentrations but not by the addition of 30 S and 50 S subunits washed with high salt concentration. GTP did not stimulate this dissociating action. 2 μg of our most purified preparation caused 100% dissociation of 100 μg of 70 S ribosomes without added GTP. DF‐induced dissociation is a very rapid process at 37°C and is temperature‐dependent in the range of 0°–37°C. DF, which is thermolabile factor, is much less or not effective with complexed 70 S ribosomes bearing peptidyl‐tRNA and mRNA.

STIMULATION AND INHIBITION OF POLYPEPTIDE SYNTHESIS BY STREPTOMYCIN IN RIBOSOMAL SYSTEMS OF ESCHERICHIA COLI, PROGRAMMED WITH VARIOUS MESSENGERS.

1. The action in vitro of streptomycin on polypeptide synthesis has been studied with cell-free extracts from streptomycin-sensitive and resistant Escherichia coli. Various messengers, such as turnip yellow mosaic virus RNA, alfalfa mosaic virus RNA, polyuridylic acid and polyadenylic acid were employed, and ribosomes still associated with their endogenous messenger(s) were also included. 2. Varying effects of streptomycin on ribosomes from streptomycin-sensitive cells with messengers of viral origin and endogenous messenger(s) have been recorded. Depending on the Mg2+ concentration either stimulation or inhibition was found. 3. Polyuridylic acid-directed polyphenylalanine synthesis and polyadenylic acid-induced polylysine formation were generally inhibited by streptomycin at all Mg2+ concentrations studied. 4. The effects exerted by streptomycin on ribosomal systems from streptomycin-resistant cells were usually small or negligible. 5. Stimulation of “sensitive” systems was exerted on a ribosomal site. 6. Streptomycin-action became apparent during protein synthesis immediately after the antibiotic was added. The degree of inhibition or stimulation was the same whether the antibiotic was added before or during the process of protein synthesis. 7. The results are discussed in relation to current concepts of streptomycin action.

The 3′‐terminus of 16 S ribosomal RNA of Escherichia coli. Isolation and purification of the terminal 49‐nucleotide fragment at a milligram scale

Recently special interest has been focussed upon the important role of the 3’4erminus of 16 S ribosomal RNA of Escherichia coli in protein synthesis. A number of proteins which are required for initiation of protein synthesis, like Sl [ 1,2], S12 [3] , IF-2 and IF-3 [4] , are located at or near the 3’-end of 16 S RNA [S-8] . Furthermore, cleavage of the 16 S RNA at a site which is 49 nucleotides from the 3’-end by colicin E3 [9] or cloacin DF13 [lo] blocks protein synthesis completely [ 111. We found it to impair the response of the ribosome to the initiation factor IF-1 [12]. Finally, it has been proposed that a sequence of nucleotides near the 3’-end of 16 S RNA base-pairs during initiation with ribosomal binding sites on messenger RNA [13-141. In order to obtain a more detailed insight in how this end of 16 S RNA is involved in protein biosynthesis, it is important to study its structure and the way in which this structure is influenced by ribosomal proteins and initiation factors. As a first approach we isolated the RNA fragment which is formed after cloacin treatment of ribosomes in large amounts in order to

Effects of heterologous ribosomal binding sites on the transcription and translation of the lacZ gene of Escherichia coli

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.

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity.

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)