We have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit... Show moreWe have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit translational activity in vivo and to affect ribosome stability in vitro, The majority of the mutant 30S particles were present as free subunits in which a reproducible decrease in amount of proteins S1, S2, S18 and S21 was observed, The protein gels also showed the appearance of a satellite band nest to S5, This band reacted with anti-S5 antibodies and had a slightly increased positive charge, The simplest interpretation of these findings, also considering published data, is that the satellite band is S5 with a non-acetylated N-terminal alanine, Underacetylation of S5 due to mutations in the 16S rRNA implies that the modification is performed on the ribosome. Show less
To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A(18) mutation in this structure element, Previously, this mutation, which... Show moreTo examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A(18) mutation in this structure element, Previously, this mutation, which changes the central base pair of helix 2, C-18-G(917) to an A(18)xG(917) mismatch, was shown to inhibit translation in vivo and a defect in initiation was suggested, Here, we find that the mutant 30S particles are impaired in forming 70S tight couples and predominantly accumulate as free 30S subunits, Formation of a 30S initiation complex, as measured by toeprinting, was almost as efficient for mutant 30S subunits, derived from the tight couple fraction, as for the wild-type control, However, the A(18) mutation has a profound effect on the overall stability of the subunit, The mutant ribosomes were inactivated by affinity chromatography and high salt treatment, due to easy loss of ribosomal proteins, Accordingly, the particles could be reactivated by partial in vitro reconstitution with 30S ribosomal proteins, Mutant 30S subunits from the free subunit fraction were already inactive upon isolation, but could also be reactivated by reconstitution, Apparently, the inactivity in initiation of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins, We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the formation of a 30S initiation complex, We suggest that the in vivo translational defect of the mutant ribosomes is caused by their inability to form 70S initiation complexes. Show less
We describe a system to isolate 30S ribosomal subunits which contain targeted mutations in their 16S rRNA. The mutations of interest should be present in so-called specialized 30S subunits which... Show moreWe describe a system to isolate 30S ribosomal subunits which contain targeted mutations in their 16S rRNA. The mutations of interest should be present in so-called specialized 30S subunits which have an anti-Shine-Dalgarno sequence that is altered from 5' ACCUCC to 5' ACACAC. These plasmid-encoded specialized 30S subunits are separated from their chromosomally encoded wild-type counterparts by affinity chromatography that exploits the different Shine-Dalgarno complementarity. An oligonucleotide complementary to the 3' end of wild-type 16S rRNA and attached to a solid phase matrix retains the wild-type 30S subunits. The flow-through of the column contains close to 100% mutant 30S subunits. Toeprinting assays demonstrate that affinity column treatment does not cause significant loss of activity of the specialized particles in initiation complex formation, whereas elongation capacity as determined by poly(Phe) synthesis is only slightly decreased. The method described offers an advantage over total reconstitution from in vitro transcribed mutant 16S rRNA since our 30S subunits contain the naturally occurring base modifications in their 16S rRNA. Show less
The genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a... Show moreThe genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a heptanucleotide slippery sequence and a downstream pseudoknot, is similar in appearance to those identified in retroviral RNAs. We have examined whether the proposed BWYV signal functions in frameshifting in three translational systems, i.c. in vitro in a reticulocyte lysate or a wheat germ extract and in vivo in E.coli. The efficiency of the signal in the eukaryotic system is low but significant, as it responds strongly to changes in either the slip sequence or the pseudoknot. In contrast, in E.coli there is hardly any response to the same changes. Replacing the slip sequence to the typical prokaryotic signal AAAAAAG yields more than 5% frameshift in E.coli. In this organism the frameshifting is highly sensitive to changes in the slip sequence but only slightly to disruption of the pseudoknot. The eukaryotic assay systems are barely sensitive to changes in either AAAAAAG or in the pseudoknot structure in this construct. We conclude that eukaryotic frameshift signals are not recognized by prokaryotes. On the other hand the typical prokaryotic slip sequence AAAAAAG does not lead to significant frameshifting in the eukaryote. In contrast to recent reports on the closely related potato leaf roll virus (PLRV) we show that the frameshifting in BWYV is pseudoknot-dependent. Show less
