Stoichiometric measurements of 30S and 50S ribosomal proteins from Escherichia coli

Hj, Weber

doi:10.1007/bf00333861

Cited by 147 publications

(69 citation statements)

References 37 publications

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“…A protein content of 40% as reported by Bayley [21] and verified in our laboratory would indicate that the total molecular weight of the 50 S and 30 S proteins should be around 760 000 and 360 000. Since the sums of the individual molecular weights of the 30 S proteins exceed the expected total molecular weight, we conclude, as did others, [19,23] for the 30 S particle ofE. coli, that the ribosomal 30 S subunit in H. cutirubrum is structurally heterogeneous.…”

Section: Resultssupporting

confidence: 85%

“…Strict adherence to stoichiometry may not be true as Weber [19] has shown that there is some heterogeneity in the E. coli 50 S subunit proteins and our unpublished data for HL20, HL21 indicates that this may be the case in H. cutirubrum as well. Our data for another very different procaryote would further strengthen Kurland's original contention that thc ribosome is structurally and functionally, a heterogeneous organelle [22,23].…”

Section: Resultsmentioning

confidence: 82%

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Acidic ribosomal proteins from the extreme halophile,Halobacterium cutirubrum

1973

FEBS Letters

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 82%

Acidic ribosomal proteins from the extreme halophile,Halobacterium cutirubrum

1973

FEBS Letters

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“…It is present in less than one copy per ribosome, while L2 is a unit protein (16). The above results plus the ease of reaction of L2 with BrAcPhe-tRNA encourage speculation that this protein may be intimately linked with the peptidyl transferase activity of the ribosome.…”

Section: Discussionmentioning

confidence: 95%

Identification of 50S Proteins at the Peptidyl-tRNA Binding Site of Escherichia coli Ribosomes

Oen

Pellegrini

Eilat

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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Bromoacetyl-phenylalanyl-tRNAPhe bound to 70S E. coli ribosomes reacts covalently with proteins of the 50S subunit. The major reactions are with proteins L2 and L27. In the presence of poly(U), 70S-bound bromoacetyl-phenylalanyl-tRNAPhe can participate in peptidebond formation with plhenylalanyl-tRNAPhe or puromycin.Most of the products of these reactions are also found covalently attached to L2 and L27. Chloramphenicol and sparsomycin markedly inhibit the peptide-bond formation. These results strongly suggest that bromoacetylphenylali.nyl-tRNAPhe can function as a normal peptidyltRNA and that the 50S proteins, L2 and L27, are located in the peptidyl-tRNA binding site. The side reactions of bromoacetyl-phenylalanyl-tRNAPhe are with one or more 50S proteins from the set L14-17, L6 and/or L11, and L26.These occur to a much less extent than the reactions with L2 and L27. Any functional significance of the side reactions is unknown.Previously we described the preparation and some of the properties of the peptidyl-tRNA analogue, N-bromoacetylphenylalanyl-tRNAPhe (BrAcPhe-tRNA). This compound was designed as a potential affinity label for the P site of Escherichia coli ribosomes (1). When bound to 70S ribosomes with poly(U) as messenger, this analogue reacted covalently with the 50S subunit. Initial studies suggested that there were two sites to which the reagent became covalently attached. BrAcPhe-tRNA was found associated with a single band in a one-dimensional polyacrylamide gel separation of the 50S proteins. This band contains proteins 3VI, 2IX, and 2XI [numbering according to Traut et al. (2)]. BrAcPhe-tRNA was also found associated with 23S rRNA. This finding implied either covalent attachment to the RNA or to a protein tightly bound to the RNA.We have now been able to define more clearly the sites of covalent attachment of BrAcPhe. Two-dimensional gel electrophoresis has been used to obtain fairly unambiguous identification of the covalently modified 50S proteins. Also, improved procedures lead to a 30-fold increase in

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“…The lack of technologies to accurately identify and quantify proteins limited most early studies of RP stoichiometries in 30S and 40S fractions of bacterial ribosomes purified from sucrose gradients [28,29]. These fractions also contained immature ribosome biogenesis particles [30][31][32] that complicated the interpretation of measured stoichiometries; furthermore, difficulties of protein identification made it hard to distinguish between core RPs and auxiliary factors [28,29]. More recently, quantitative mass-spectrometry (MS) has begun to provide direct evidence for differential RP synthesis [33] and differential RP stoichiometry in isolated ribosomes [23].…”

Section: The Existence Of Eukaryotic Ribosomes With Distinct Ribosomamentioning

confidence: 99%

“…The biochemical evidence for specialized ribosomes fulfilling physiological roles in wildtype cells had until recently remained indirect, mostly limited to differential RP-transcript levels. The lack of technologies to accurately identify and quantify proteins limited most early studies of RP stoichiometries in 30S and 40S fractions of bacterial ribosomes purified from sucrose gradients [28,29]. These fractions also contained immature ribosome biogenesis particles [30][31][32] that complicated the interpretation of measured stoichiometries; furthermore, difficulties of protein identification made it hard to distinguish between core RPs and auxiliary factors [28,29].…”

mentioning

confidence: 99%

Ribosome stoichiometry: from form to function.

Emmott

Jovanović

Slavov

2018

Preprint

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The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis been speculated for over sixty years. Recent advances in mass spectrometry and high throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast or mammalian systems. In addition to RP stoichiometry, ribosomes play host to a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible these proteoforms combine to function as a 'ribosome code' to tune protein synthesis? We outline the specific benefits that translational regulation by specialized ribosomes can offer and discuss the means and methodologies available to correlate and characterize RP stoichiometry with function. We highlight previous research with a focus on formulating hypotheses that can guide future experiments and crack the 'ribosome code'.Ribosomes, the cellular machinery of protein synthesis, are present at up to ten million copies per cell in mammals. Despite their abundance and the wide array of known modifications to both ribosomal proteins (RPs) and rRNA, study of the direct role of the ribosome in tuning cellular translation has until recently taken a back seat to posttranscriptional regulation at the level of translation initiation. The hypothesis that ribosomes actively regulate protein synthesis as part of normal development and physiology dates back to the 1950s [1]. In the ensuing decades, numerous albeit inconclusive, observations have supported this hypothesis, and a subset of those are shown in Figure 1, color-coded by the type of evidence.For many years, the dominant 'abundance' model of translational regulation by the ribosome suggested a limited role for ribosomes in translation regulation [2]. In this model, if ribosomes have different initiation affinity for different transcripts, a global decrease in the availability of free ribosomes selectively decreases the initiation rates of different transcripts to varying degrees [2]. This mechanism, recently reviewed by Mills and Green [4], relies on the non-linear dependence of translation initiation on free ribosomes and applies quite generally. Indeed, recent research from the Sankaran lab suggested this mechanisms could explain the failure of erythroid lineage commitment seen with Diamond-Blackfan anaemia [5] 2 by the abundance model is limited in magnitude by the changes of total ribosomal content and in flexibility since it provides unidirectional regulation for all proteins.The concept of ribosome specialization In the 'specialized' ribosome model, ribosomes do not possess constant structure or composition (Figure 2A, B), and instead exhibit altered stoichiometry of what were previously thought to represent 'core' ribosomal proteins (Figure 1) [7,8]. In this model, different ribosomal compositions are functional and have specific roles in translation. Specialized ribosomes could co-exist within cells, or between ...

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Stoichiometric measurements of 30S and 50S ribosomal proteins from Escherichia coli

Cited by 147 publications

References 37 publications

Acidic ribosomal proteins from the extreme halophile,Halobacterium cutirubrum

Acidic ribosomal proteins from the extreme halophile,Halobacterium cutirubrum

Identification of 50S Proteins at the Peptidyl-tRNA Binding Site of Escherichia coli Ribosomes

Ribosome stoichiometry: from form to function.

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