The ribosomal protein S4 is a translational repressor that binds to a complex mRNA pseudoknot structure containing the ribosome binding site for the first gene of the a operon. Either 30S subunits or S4 protein bound to the mRNA causes Moloney murine leukemia virus reverse transcriptase to pause near the 3' terminus of the pseudoknot. There is no competition between subunits and S4 for mRNA binding. The kinetics of forming S4-30S-mRNA complexes are biphasic, and the fraction of mRNA molecules reacting more rapidly decreases as the temperature is increased from 30°C to 40°C. A number of genes are regulated at the translational level by repressor proteins that bind mRNA and inhibit translation. The first repressor-mRNA complex observed was between the phage R17 replicase RNA and the phage coat protein (1). Since then, a number of other prokaryotic translational repressors have been identified (2-4). Among them are a set of ribosomal proteins that regulate the translation of most ribosomal protein operons in Escherichia coli (5), a substantial fraction of the total protein synthesis in the cell.Ribosomal protein S4 binds to a single target site in the a operon mRNA and represses the translation of all four ribosomal proteins in the operon (6). The target structure recognized by S4 and required for translational repression is a complex pseudoknot that encompasses the ribosome binding site (7). Comparison of in vitro S4 binding data and in vivo measurements of translational repression with a series of mRNA mutants gave the unexpected result that some mutants were able to bind S4 with approximately wild-type affinities but showed substantially reduced S4-mediated repression levels (8). This result can be explained if S4 is an allosteric inhibitor of translation: the model proposes that the ribosome and S4 repressor bind to separate sites on the mRNA and that S4 binding traps the mRNA in a conformation unable to form a competent initiation complex. We have presented (9) evidence for the existence of "active" and "inactive" mRNA conformations required by this model.In this paper we examine the effects of S4 protein on initiation complex formation and find support for the allosteric mechanism of translational repression. The protein apparently traps the mRNA in a conformation able to bind 30S subunits but unable to bind tRNAf et stably. for 15 min were carried out as described (9), except that S4 buffer (60 mM NH4C1/35 mM KCI/10 mM Tris acetate, pH 7.4/10 mM magnesium acetate/6 mM 2-mercaptoethanol/50 mM urea) was used. MATERIALS AND METHODSThe strengths of toeprint signals were quantitated from autoradiograms by densitometry (9). The "relative toeprint" is the intensity of the toeprint bands divided by the sum of the toeprint and full-length transcript intensities. RESULTS30S Subunit-Dependent Toeprint Signals with the a mRNA.Hartz et al. (11) have shown that the ternary tRNAfj tt-30S subunit-mRNA initiation complex inhibits transcription by reverse transcriptases. The prematurely terminated transcripts, which g...
In this study we present a ninhydrin based microwell assay that can be utilized in place of the traditional Kjeldahl method for the determination of the protein content of beer or wine. In addition, the assay is ideal for the determination of free amino acids in beer (FAN), a term understood and used by brewers, and yeast assimilable nitrogen (YAN) used by enologists. The assay only measures alpha amino acids and ammonia so other nitrogen sources are not detected, resulting in a 30% reduction in total protein of a variety of beers compared to the Kjeldahl method, which measures nitrogen from all sources. The results also showed that only 25% of the total "protein" in beer is actually derived from peptides larger than 3,500 Kd. Analysis of beer or wine with the microwell assay for total usable nitrogen was compared to the standard FAN and YAN methods and conditions were determined for maximal efficiency and precision. Superior results were obtained with low reaction volumes and a stable sodium acetate buffered ninhydrin reagent at pH 5.5. As an alternative, for use with cuvettes, a reduced volume FAN assay using the same pH 5.5 sodium acetate buffered ninhydrin reagent gave comparable results. The assay is economical, rapid, accurate and applicable to large numbers of samples.
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