Peter H. van Knippenberg

Site-specific mutation of the conserved m62 A m62 A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase

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.

Optimized bacterial production of nonglycosylated human transferrin and its half-molecules

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.

Polyuridylic acid-dependent binding of fMet-tRNA to Escherichia coli ribosomes and incorporation of formylmethionine into polyphenylalanine.

It is generally accepted that initiat~o~i of protein synthesis in Eschcrichia coli starts with N-formylmethionine, directed by the codons AUG or GUG. ln one case, reinitiation on the mRNA of amber mutants of luc repressor, ULJG is used as the irlitiation codon [Il. Early studies [2,3] indeed showed that the triplets AUG, GUG and LJUG are the most effective in stimulating diet-tRNA binding to ribosomes in vitro. Synthetic polynucleotides containing AUG and/or GUG codons as well as natural mRNA have been used extensively in order to elucidate the mechanism of initiation of protein synthesis (reviewed in 141). In all these studies it has been assumed that binding of fMet-tRNA to ribosomes is directed by AUG or GUG codons present in the polynucleotide. This paper describes a study in which poly(U) is used to direct fMet-tRNA binding to 30 S ribosomes. The f&let is incorporated into polyphenylalanine when 50 S subunits and other factors for protein synthesis are added.

Polyribosomes of Escherichia coli

SummaryThe distribution of pulse labeled RNA, pulse labeled protein, soluble enzyme (β-galactosidase) and polyribosomes between low speed (10 min 20000xg) supernatant and pellet of E. coli lysates was examined. This distribution was greatly changed by addition of deoxyribonuclease to the lysing medium. Large amounts of polysomes sedimented with DNA at low centrifugal forces. A complex of membrane, DNA, polyribosomes and RNA polymerase could be separated from unlysed cells by surcrose gradient centrifugation. The polysomes present in this complex (Polysomes II) were separated from the polysomes which were found in the “cytoplasma” (Polysomes I). Polysomes II contain very few ribosomal subunits and 70S ribosomes.

Increased kasugamycin sensitivity in Escherichia coli caused by the presence of an inducible erythromycin resistance (erm) gene of Streptococcus pyogenes

SummaryAn 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 μg/ml to>3000 μg/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 dimenthylation.

Biochemical characterization of ribosomes of kasugamycin-dependent mutants of Escherichia coli

The ribosomal initiation process can be investigated using the antibiotic kasugamycin (Ksg), which inhibits the binding of fMet-tRNAf to the ribosome [l]. Strains resistant to Ksg due to a mutation at the Q&4 * locus [2 3 have lost the activity of a methylase which acts on two adjacent adenosines near the 3’-end of the 16 S RNA [3 1. Though resistance is a function of the 30 S subunit, it is only manifested in the presence of 50 S subu~ts [4], ~ethylation of these adenosines near the 3’end of the 16 S is apparently universal to all organisms, but its functional significance is not known for certain. The presence of the methyl groups facilitates subunit association [5]. As an additional probe of this ribosomal neighbourhood, Ksgdependent mutants have been isolated and characterized [6]. A number of these mutants are I&resistant as well as dependent, yet (as found by genetic analysis) do not have a KsgA mutation. Mutational alterations in 50 S proteins have been shown in some of the mut~ts to be responsible for the mut~t phenotype [6,7], possibly an in vivo indication of the above-cited in vitro effect of 50 S subunits on 30 S phenotype [4]. It was considered worthwhile to analyze the ribosomes of these Ksg-dependent mutants in vitro. The mutants selected had a variety of phenotypes, and were tested for the methylation of the 16 S RNA, and. for presence or absence of methylase. The effect of&g on fMet-tRNAf binding and on a natural message-directed polypeptide synthesizing system was also determined, to see if Ksg-dependence could be demonstrated in vitro.