The sequence of the 3'-terminus of 16S RNA from different bacteria has been determined. Complementarity relationships between this sequence and a purine-rich tract in the ribosome binding site of different bacterial mRNAs suggest that the 3'-end of 16S RNA determines the intrinsic capacity of ribosomes to translate a particular cistron.

Determinant of cistron specificity in bacterial ribosomes

https://mmbr.asm.org/content/mmbr/47/1/1.full.pdf

Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.

RNA chaperones and the RNA folding problem.

https://userpages.umbc.edu/~farabaug/lab/papers/annrevgen1996.pdf

Programmed translational frameshifting.

Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by approximately 6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components.

/pdf/trna-dynamics-on-the-ribosome-during-translation-1s4x58h0e1.pdf

tRNA dynamics on the ribosome during translation

The previously reported requirement of ribosomal protein S1 for translation of phage RNA is now shown to be related to the involvement of the protein in initiation complex formation. The structure of the messenger RNA appears to be uniquely related to S1 function, since translation and initiation with mildly unfolded phage RNA (by modification with formaldehyde) are independent of S1. It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA. A model is suggested for S1 function in both initiation of protein synthesis and initiation of phage RNA replication.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10330.x

The Specific Role of Ribosomal Protein S1 in the Recognition of Native Phage RNA

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

1
The binding of washed 30-S ribosomal subunits of Escherichia coli to MS2 RNA in the absence of fMet-tRNA requires the presence of the initiation factor IF-3. No binding is observed with IF-1, IF-2 or a combination of these two initiation factors.

2
The IF-3-dependent binding is stimulated about two-fold by IF-2. IF-1 has no effect in this respect. Optimal binding occurs at about 7 mM Mg2+.

3
Upon incubation of [35S]IF-3, MS2 [3H]RNA and 30-S subunits, complexes are formed which contain the three components in a 1:1:1 ratio. These ternary complexes have a sedimentation coefficient of about 40 S and a buoyant density in CsCl of about 1.74 g/ml.

4
The ternary complexes formed in the absence of IF-2 and IF-1 are rather labile and readily dissociate into MS2 RNA and IF-3-containing ribosomes. Their half-life at 0°C is about 40 min. In the presence of IF-2 and IF-1, complexes are formed which remain stable for at least 6 h. Unwashed native 30-S subunits also form stable complexes with MS2 RNA.

5
Binding of washed 30-S subunits to unfolded MS2 RNA (MS2 RNA treated with formaldehyde) does not require initiation factors. Complexes containing more than one ribosomal particle per messenger can be formed. Attachment of fMet-tRNA to these complexes requires IF-2 and IF-1, but is optimal in the presence of all three initiation factors.

6
Release of [35S]IF-3 from the MS2 RNA · 30-S subunit complexes occurs upon attachment of fMet-tRNA. In the case of unwashed native 30-S ribosomal subunits, recycling of IF-3 occurs at the same stage as was demonstrated by a direct determination of the stoichiometry of unlabeled IF-3 on the ribosomal complexes.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb03197.x

Initiation Factor‐Dependent Binding of MS2 RNA to 30‐S Ribosomes and the Recycling of IF‐3

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

The ability of 24 systematically modified analogues of adenosine 3′,5′-monophosphate (cAMP) to enhance the synthesis of β-galactosidase in glucose-repressed Escherichia coli strains KNBL 1001 and cpd– Crookes has been investigated. The properties of the analogues in comparison with cAMP are, with only two exceptions, alike in both strains. Two analogues, 7-deazaadenosine 3′,5′-monophosphate (i.e. tubercidin 3′,5′-monophosphate) and (Rp)-adenosine 3′,5′-monothionophosphate, exhibit higher biological activity than cAMP. The latter analogue is 50-fold more active in both strains. Three analogues showed activities comparable to cAMP, four analogues were less active and 12 analogues were unable to antagonize catabolite repression. Structure-activity correlations showed that the 2′OH-, 3′O-, 5′O-, the negative charge and the 6-amino group cannot be modified without losing biological activity in vivo, while the N-1 and N-7 in adenine are not essential. The interaction with the catabolite gene activator protein is stereoselective for an unmodified axial exocyclic oxygen. The results are compared to those obtained with cAMP analogues in E. coli in vitro and those obtained with the same analogues in protein-kinase systems and Dictyostelium species. The model of McKay et al. [McKay, D. B., Weber, J. T. and Steitz, T. A. (1982) J. Biol. Chem. 257, 9518–9524] proposed for distinct chemical interactions of cAMP with the catabolite gene activator protein is discussed and supplemented by additional hydrogen bond interactions.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1984.tb07887.x

Investigations on stimulation of lac transcription in vivo in Escherichia coli by cAMP analogues. Biological activities and structure-activity correlations.

Peter H. van Knippenberg

Papers

The Specific Role of Ribosomal Protein S1 in the Recognition of Native Phage RNA

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

Initiation Factor‐Dependent Binding of MS2 RNA to 30‐S Ribosomes and the Recycling of IF‐3

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

Investigations on stimulation of lac transcription in vivo in Escherichia coli by cAMP analogues. Biological activities and structure-activity correlations.