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At least 19 recordsLinked to original sources
PubMed1983

A method for mapping RNA initiation, termination, splice, and protein binding sites. Ribosome binding sites on beta-globin messenger RNA.

A new method for mapping RNA initiation, termination, and splice sites was developed. The method involves: 1) hybridization of RNA to end-labeled single-stranded DNA; 2) mild digestion of the hybrid with a single strand specific nuclease; 3) high resolution gel electrophoresis and autoradiography. The regions of the labeled probe which are resistant to nuclease digestion are mapped by measuring the distance from the labeled end. The sequence can be determined by running the end-labeled probe sequenced according to the protocol of Maxam and Gilbert (Maxam, A.M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) at the same time. The feasibility of this method was tested with adult chicken beta-globin mRNA, and we found that the transcription initiation, termination, and RNA splice sites can be determined to within a few bases of the known sites. Using this method we also found that ribosomes bind to beta-globin mRNA from 22 bases upstream from the translation start codon (AUG) to 15 bases past the stop codon (UAA).

Animals↗
PubMed1989

Mapping the lacZ ribosome binding site by RNA footprinting.

The ribosome binding site of the Escherichia coli lacZ mRNA has been characterized by using an RNA footprinting technique. Purified E. coli 70S ribosomes and fMet-tRNA were incubated with mRNA, and the complex was treated with RNA-reactive reagents or RNases as probes. The protected sites on the mRNA were then mapped by extending a radioactive primer with reverse transcriptase. Dimethyl sulfate, diethyl pyrocarbonate, and 1,10-phenanthroline-copper ion oxidative complex were used as reagent probes; they detected interaction sites within the ribosome binding site. A region of approximately 35 nucleotides was protected by the ribosome, specifically across the Shine-Dalgarno region, around the fMet initiation codon, and at a region 7-12 nucleotides distal to the fMet codon. In addition, an enhanced reaction occurred between the fMet codon and the distal site. These results imply an internally selective interaction between the ribosome and the mRNA sequence. The enhanced reactivity of a site distal to the initiation site--flanked by the AUG codon and a site previously identified as conserved in a study of initiation sequences--may indicate a region where the mRNA is specifically exposed.

Alkylating Agents↗
PubMed1980

The ribosome binding sites recognized by E. coli ribosomes have regions with signal character in both the leader and protein coding segments.

Oligonucleotide analysis, by a novel computerized procedure, was first applied to determine the sequence of an ideal E. coli promoter (Scherer et al., Nucl. Acids Res. 1978, 5:3759-3773) and has now been used to obtain the sequence of nucleotides that should be present in a messenger RNA for optimum binding to the E. coli ribosome. This sequence is: UU.UUAAAAAUUAAGGAGGUAUAUUAUGAAAAAAAUUAAAAAACUCAA AA U A AUA A CUC G. Comparison of this sequence with each of the 68 ribosome binding site sequences used to generate it shows a preference rather than an absolute requirement for a specific base in any given position. The preference for certain bases persists along the whole length of the RNA within the ribosome binding domain even though nearly half of that length includes translated codons. Thus messages without leader sequences (like lambda CI mRNA) can still have some affinity for the ribosome. Part of the model sequence is complementary to the 3'end of 16S rRNA.

Base Sequence↗
PubMed1980

Location of ribosome-binding sites in the nin5 region of bacteriophage lambda.

Seven ribosome-binding sites on DNA have been located within the region defined by the nin5 deletion as well as several ribosome-binding sites on each side of the nin5 region. These were mapped by electron microscopy relative to the end points of the nin5 deletion and two Tn903 transposons, one inserted into gene Rz and another inserted near gene Q. These ribosomes binding sites within the nin5 region may correspond to polypeptide initiation sites for up to seven new dispensible lambda genes.

Bacteriophage lambda↗
PubMed1993

Nature of the ribosomal mRNA track: analysis of ribosome-binding sites containing different sequences and secondary structures.

The ribosomal mRNA track was investigated by toeprinting 30S ribosomes, in the presence or absence of tRNA, using a variety of different ribosome-binding sites. We found that: (1) the ribosome, by itself, recognizes the mRNA translational initiation site; (2) the ribosomal mRNA track makes extensive contact with mRNA independent of tRNA and the start codon; (3) ribosome-mRNA complexes are less stable than complexes containing tRNA; and (4) toeprinting can be used to analyze the contour of the ribosomal mRNA track, yielding information on its "height" as well as its "length" dimension. Examination of several ribosome-binding sites, including those containing very stable secondary structure, indicated that the "height" of the mRNA track is quite roomy, while the nucleotide distance between the site of Shine-Dalgarno annealing, the P site, and the 3'-edge of the mRNA track is fixed. The data suggest a mechanism for tethering regulatory elements to the ribosome during translation.

Base Sequence↗
PubMed1978

Location of ribosome-binding sites on the tetracycline resistance transposon Tn10.

Eight ribosome-binding sites were located on the single-stranded Tn10 DNA loop which was formed after denaturation of lambda phage DNA containing the Tn10 transposon sequence. Ribosomes were bound only to the Tn10 loop contained on the R strand of lambda DNA but not to that on the L strand, suggesting that one of the two strands of Tn10 DNA is selectively transcribed. Six of the eight ribosome binding sites were located in one-half of the DNA loop. The maximum sizes of potential polypeptides were calculated for these genes to range between 9,500 and 84,000 daltons.

Binding Sites↗
PubMed1987

More than 150 nucleotides flanking the initiation codon contribute to the efficiency of the ribosomal binding site from bacteriophage T7 gene 1.

The ribosomal binding site (RBS) from gene 1 of bacteriophage T7 was isolated on fragments of differing length and cloned upstream of the mouse dihydrofolate reductase gene to control the translation of its sequence. A 29 base pair sequence containing all elements generally believed to be essential for the RBS's showed extremely low activity. Additional upstream and downstream sequences were required to obtain a several orders of magnitude higher efficiency. By contrast, areas further downstream than +112 nucleotides from the initiator proved to be inhibitory, whereas the presence of an upstream RNaseIII cleavage site showed a strong stimulatory effect. This suggests that tertiary structures are involved in the function of the RBS studied. The efficient RBS's were complexed by ribosomes at much lower concentrations of the mRNA than the weak ones.

Base Sequence↗
PubMed1999

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Amino Acid Sequence↗
PubMed1994

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.

Base Sequence↗
PubMed1994

Ribosome binding site consensus sequence of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H.

The putative ribosome binding sites preceding 32 of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H genes were compared. A highly conserved consensus sequence for the ribosome binding sites of LL-H genes was inferred, GAAAGGAG. This study included the characterization of the last nucleotides of the 3'-end of the 16S rRNA molecule from L. delbrueckii subsp. lactis and its comparison to the ribosome binding site consensus sequence.

Bacteriophages↗
PubMed1967

The ribosomal binding site for peptidyl-transfer-ribonucleic acid.

The properties of a site, on Escherichia coli ribosomes, which binds peptidyl-s-RNA (where s-RNA refers to ;soluble' or transfer RNA) have been investigated. The binding is stable both in low Mg(2+) concentration (0.1mm), and in high Mg(2+) concentration (10mm) in the absence or presence of potassium chloride (86mm). Puromycin has been used to break the bond between the s-RNA and the polypeptide, and in the absence of further protein synthesis this technique exposes free s-RNA molecules on the ribosomes. The s-RNA exposed remains bound in high Mg(2+) concentration, but the binding is unstable in high Mg(2+) concentration with potassium chloride and the s-RNA can be freed completely from the ribosomes by lowering the Mg(2+) concentration. It can also be displaced by s-RNA in the medium. It is suggested that this ribosomal binding site for peptidyl-s-RNA is the site for peptide bond formation. Further, it is proposed that it is the same site that can be demonstrated on ribosomes not engaged in protein synthesis and that, in high Mg(2+) concentration, will bind s-RNA molecules charged or uncharged with amino acids.

Binding Sites↗
PubMed1989

In vivo selected promoter and ribosome binding site up-mutations: demonstration that the Escherichia coli bla promoter and a Shine-Dalgarno region with low complementarity to the 16 S ribosomal RNA function in Bacillus subtilis.

We have constructed a plasmid, pQS1, in which a mouse dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP:oxidoreductase; EC 1.5.1.3; DHFR) cDNA is inserted in the unique PstI site of a gram-positive/gram-negative shuttle vector derived from pBR322. The cDNA is expressed under the control of the bla promoter, which, like most gram-negative bacterial genes, is considered not to be expressed in Bacillus subtilis, and its coding sequence is translated from a polycistronic message. We have selected in vivo and studied, in Escherichia coli and B. subtilis, expression mutants with promoter and ribosome binding site sequence mutations. One promoter mutation changes the third nucleotide of the -35 region from a C to a G. As expected, this substitution results in increased transcriptional activity in E. coli. In B. subtilis, this mutation induces the accumulation not only of a low but significant amount of dhfr mRNA but also of DHFR, demonstrating that binding strengths with a free energy as low as -9.4 kcal/mol are sufficient to promote ribosome binding in B. subtilis. The association of the promoter mutation (C-G) with a mutation which creates a strong B. subtilis ribosome binding site (-21 kcal/mol) results in the accumulation of a large amount of dhfr mRNA. This demonstrates the importance of having an efficient ribosome binding site in the evaluation of promoter function: for example, with this strong ribosome binding site we can show that the wild-type bla promoter is recognized by the B. subtilis transcription machinery.

Animals↗
PubMed1983

Translation of insulin-related polypeptides from messenger RNAs with tandemly reiterated copies of the ribosome binding site.

Plasmids have been constructed containing reiterated copies of a 66 bp fragment, loosely referred to as the ribosome binding site, that includes the AUG initiator codon of preproinsulin. The extreme test involved plasmid 255/17, which carried four tandem copies of the ribosome binding site, with all four AUG triplets in the same reading frame as the preproinsulin coding sequence downstream. Initiation at any potential start site would generate a polypeptide precipitable with anti-insulin antiserum, and its size would reveal the AUG(s) active in initiation. One insulin-related polypeptide was synthesized in cells transfected by p255/17; its size corresponded to the product initiated at the first ribosome binding site in the tandem array. Inasmuch as the three downstream AUG triplets, which are not used, occur in a sequence context identical with that around the 5'-proximal AUG triplet, which is used, the position of an AUG triplet relative to the 5' end of the mRNA appears to be important in identifying it as a functional initiator codon.

Base Sequence↗
PubMed1981

Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.

With the premise that mRNAs transcribed in Escherichia coli from cloned eukaryotic DNA inserts do not possess the necessary regulatory signals for recognition by prokaryotic ribosomes, we have constructed a general plasmid vector carrying a chemically synthesized prokaryotic ribosome binding site that will ensure the efficient expression of eukaryotic proteins in E. coli. In addition to the regulatory signals necessary for ribosome recognition, the synthetic segment contains, at one end, a Pst I cleavage site which will direct its insertion to pBR322 DNA and, at the other end, a HindIII site to facilitate attachment of the passenger eukaryotic gene. Using simian virus 40 (SV40) tumor (t) antigen as a model system, we have ligated the SV40 DNA fragment containing the entire t antigen gene in tandem with the synthetic ribosome binding site to pBR322 DNA at the Pst I site, which lies within the coding sequence of the beta-lactamase gene. Initiation of transcription at the beta-lactamase promoter would produce a chimeric mRNA with the synthetic ribosome binding signals and the SV40 sequence flanked by beta-lactamase coding sequences. Utilization of the synthetic regulatory signals for initiation of translation is demonstrated by the efficient synthesis, in bacterial transformants, of authentic SV40 t antigen. Excision of the entire SV40 insert by HindIII from those clones that have retained intact HindIII sites at the junction between the ribosome binding site and the SV40 sequence would allow insertion of other heterologous DNAs by using HindIII linkers. The efficient expression of any DNA insert would require that the entire coding sequence be contiguous and that its termini be randomized by treatment with exonuclease III and nuclease S1 to vary the distance between the translational initiation codon and the synthetic ribosome binding site.

Antigens, Neoplasm↗
PubMedDate not supplied

Antisense RNA: effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules.

A series of plasmids were constructed to generate RNA complementary to the beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5' and/or the 3' region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of beta-galactosidase synthesis occurred when a functional ribosome binding site was present near the 5' end of the anti-mRNA and the anti-mRNA synthesized was complementary to the 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA:normal lacZ mRNA ratios. A functional ribosome binding site at the 5'-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorporation of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of beta-galactosidase synthesis.

Binding Sites↗
PubMed1984

Identification of a ribosome-binding site for a leader peptide encoded by Rous sarcoma virus RNA.

A new method for identifying ribosome-binding sites was developed to determine whether AUG codons in the 5'-terminal RNA sequence of Rous sarcoma virus were used to initiate protein synthesis. We found that when translation is inhibited, the major ribosome-binding site on Rous sarcoma virus RNA is at the 5'-proximal AUG codon, even though the primary translational product from this RNA, Pr76gag, is encoded behind the fourth AUG codon 331 bases downstream from the observed initiation site. These results suggest that ribosomes can initiate translation on Rous sarcoma virus RNA at more than one site, thereby producing a seven-amino-acid peptide, as well as the gag gene polyprotein precursor of Mr 76,000.

Animals↗