Quantitative analysis of ribosome binding sites in E.coli.

185 clones with randomized ribosome binding sites, from position -11 to 0 preceding the coding region of beta-galactosidase, were selected and sequenced. The translational yield of each clone was determined; they varied by more than 3000-fold. Multiple linear regression analysis was used to determine the contribution to translation initiation activity of each base at each position. Features known to be important for translation initiation, such as the initiation codon, the Shine/Dalgarno sequence, the identity of the base at position -3 and the occurrence of alternative ATGs, are all found to be important quantitatively for activity. No other features are found to be of general significance, although the effects of secondary structure can be seen as outliers. A comparison to a large number of natural E.coli translation initiation sites shows the information profile to be qualitatively similar although differing quantitatively. This is probably due to the selection for good translation initiation sites in the natural set compared to the low average activity of the randomized set.     

The interaction of E. coli IHF protein with its specific binding sites

We have used two kinds of footprinting techniques, dimethylsulfate interference and hydroxyl radical protection, to explore the way that IHF recognizes its specific target sequences. Our results lead us to conclude that IHF recognizes DNA primarily through contacts with the minor groove, an unprecedented mode for a sequence-specific binding protein. We have also determined that, although IHF is a small protein that protects a large region of DNA, only a single IHF protomer is present at each binding site. IHF bends the DNA to which it binds. We have combined this fact plus our footprinting and stoichiometry data together with the crystal structure of a related protein, the nonspecific DNA binding protein HU, to propose a model for the way in which IHF binds to its DNA target.     

Simultaneous binding of trigger factor and signal recognition particle to the E. coli ribosome

Signal recognition particle (SRP) and trigger factor (TF) both bind to ribosomal protein L23 at the peptide exit area on the 50S subunit of the E. coli ribosome. In this study, we have developed a spin-down assay and used it to estimate KD values and the corresponding enthalpies for the binding of radio-labelled SRP and TF to naked ribosomes and to ribosomes carrying a tetrapeptidyl-tRNA in the P site. At 20 degrees C, the KD value for TF binding is 2 microM and for SRP it is 170 nM for naked as well as for translating ribosomes. At 4 degrees C, the KD values for TF and SRP binding are 1.1 microM and 90 nM, respectively. Competition binding experiments reveal that SRP and TF bind simultaneously to the ribosome with little affinity interference, showing that the factors have separate binding sites on L23. This makes an alternating binding mode for TF and SRP less plausible.     

Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE

The direct assays on Biacore with immobilised RRF and purified L11 from E. coli in the flow trough have shown unspecific binding between the both proteins. The interaction of RRF with GTPase domain of E. coli ribosomes, a functionally active complex of L11 with 23S r RNA and L10.(L7/L12)₄ was studied by Biacore. In the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E. coli were used the antibiotics thiostrepton, tetracycline and neomycin and factors, influencing the 70S dissociation Mg²⁺, NH₄Cl, EDTA. The binding is strongly dependent from the concentrations of RRF, Mg²⁺, NH₄Cl, EDTA and is inhibited by thiostrepton. The effect is most specific for 50S subunits and indicates that the GTPase centre can be considered as a possible site of interaction of RRF with the ribosome. We can consider an electrostatic character of the interactions with most probable candidate 16S and 23S r RNA at the interface of 30S and 50S ribosomal subunits.     

Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins--insight into the mechanism of the ribosome recycling step

Ribosome recycling factor (RRF), elongation factor-G (EF-G), and ribosomes from Thermus thermophilus (tt-) and Escherichia coli (ec-) were used to study the disassembly mechanism of post-termination ribosomal complexes by these factors. With tt-RRF, ec-EF-G can release bound-tRNA from ec-model post-termination complexes. However, tt-RRF is not released by ec-EF-G from ec-ribosomes. This complex with tt-RRF and ec-ribosomes after the tRNA release by ec-EF-G is regarded as an intermediate of the disassembly reaction. Not only tt-RRF, but also mRNA, cannot be released from ec-ribosomes by tt-RRF and ec-EF-G. These data suggest that the release of RRF from ribosomes is coupled or closely related to the release of mRNA during disassembly of post-termination complexes. With tt-ribosomes, ec-EF-G cannot release ribosome-bound ec-RRF even though they are from the same species, showing that proper interaction of ec-RRF and ec-EF-G does not occur on tt-ribosomes. On the other hand, in contrast to a published report, tt-EF-G functions with ec-RRF to disassemble ec-post-termination complexes. In support of this finding, tt-EF-G translocates peptidyl tRNA on ec-ribosomes and catalyzes ec-ribosome-dependent GTPase, showing that tt-EF-G has in vitro translocation activity with ec-ribosomes. Since tt-EF-G with ec-RRF can release tRNA from ec-post-termination complexes, the data are consistent with the hypothesis that the release of tRNA by RRF and EF-G from post-termination complexes is a result of a translocation-like activity of EF-G on RRF.     

Interaction between the erythromycin and chloramphenicol binding sites on the Escherichica coli ribosome.

The effects of chloramphenical on the binding kinetics of a fluorescein isothiocyanate derivative of 9(S)-erythromycylamine with 70S and 50S ribosomes have been studied by direct fluorimetric measurements. While chloramphenicol had little effect on the second-order 70S binding rate of the erythromycin analogue, it substantially reduced the dissociation rate of the fluorescent antibiotic-70S ribosome complex. This could be explained by simultaneous binding of both antibiotics to the 70S ribosome. The kinetic results suggest that chloramphenicol-saturated 70S particles bind the erythromycin analogue four times stronger and this was confirmed by direct binding studies. In additon, chloramphenicol causes a twofold increase in the intrinsic fluorescence of the 70S-bound analogue. This increase in fluorescence was used to study the kinetics of chloramphenicol binding to 70S ribosomes containing the fluorescent derivative. The fluorescence change followed first-order kinetics, suggesting that chloramphenicol induces a conformational change in the 70S particle. This could explain both its effect on erythromycin binding and on the fluorescence of bound analogue. Less detailed results with the 50S particle indicate a qualitively similar picture of erythromycin-chloramphenicol interactions.     

Studies on the RNA and protein binding sites of the E. coli ribosomal protein L10.

We have used modification of specific amino acid residues in the E. coli ribosomal protein L10 as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of L10 was found to be inhibited by modification of one more of its arginine residues. This treatment does not affect the ability of L10 to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that L10's role in promoting the L7/L12-ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously.     

Palindromic units from E. coli as binding sites for a chromoid-associated protein

Several hundred copies of a highly conserved extragenic palindromic sequence, 20-40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid-associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.     

Identification of ribosome binding sites in Escherichia coli using neural network models.

This study investigated the use of neural networks in the identification of Escherichia coli ribosome binding sites. The recognition of these sites based on primary sequence data is difficult due to the multiple determinants that define them. Additionally, secondary structure plays a significant role in the determination of the site and this information is difficult to include in the models. Efforts to solve this problem have so far yielded poor results. A new compilation of E. coli ribosome binding sites was generated for this study. Feedforward backpropagation networks were applied to their identification. Perceptrons were also applied, since they have been the previous best method since 1982. Evaluation of performance for all the neural networks and perceptrons was determined by ROC analysis. The neural network provided significant improvement in the recognition of these sites when compared with the previous best method, finding less than half the number of false positives when both models were adjusted to find an equal number of actual sites. The best neural network used an input window of 101 nucleotides and a single hidden layer of 9 units. Both the neural network and the perceptron trained on the new compilation performed better than the original perceptron published by Stormo et al. in 1982.     

Protein synthesis and competitive ESR binding studies with E. coli ribosomes and spin-labeled polynucleotides.

Enzymatically prepared spin labeled copolymers of (U)n were tested for their ability to direct polyphenylalanine synthesis in vitro using E. coli B enzymes and ribosomes. Spin labeling of the C5 position using (RUGT,U)n (1:100) or (RUTT,U)n (1:100) did not alter the amount of polyphenylalanine formed in comparison to (U)n. In contrast, the C4 spin labeled copolymer (ls4U,U)n (1:100) reduced phenylalanine incorporation by 70-75% of the (U)n control levels. ESR monitoring of competitive ribosome binding to equimolar mixtures of polynucleotides was demonstrated with the macromolecular probe (DUTT,dT)n (1:100), the DNA analogue of (RUTT,U)n. The ESR competition approach showed that the affinity of the ribosomes was essentially the same for (dT)n, (A,U,G)n, and (A,U,G)n + tRNArmet.     

Recognition sites for a membrane-derived DNA binding protein preparation in the E. coli replication origin

Summary The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is locatd in the minimum oriC (35–270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3OH-5P in the direction of the E. coli genetic map is recognized, at the second site the 5P-3OH strand.