Escherichia coli ribosomal protein L20 binds as a single monomer to its own mRNA bearing two potential binding sites

Ribosomal protein L20 is crucial for the assembly of the large ribosomal subunit and represses the translation of its own mRNA. L20 mRNA carries two L20-binding sites, the first folding into a pseudoknot and the second into an imperfect stem and loop. These two sites and the L20-binding site on 23S ribosomal RNA are recognized similarly using a single RNA-binding site located on one face of L20. In this work, using gel filtration and fluorescence cross-correlation spectroscopy (FCCS) experiments, we first exclude the possibility that L20 forms a dimer, which would allow each monomer to bind one site of the mRNA. Secondly we show, using affinity purification and FCCS experiments, that only one molecule of L20 binds to the L20 mRNA despite the presence of two potential binding sites. Thirdly, using RNA chemical probing, we show that the two L20-binding sites are in interaction. This interaction provides an explanation for the single occupancy of the mRNA. The two interacting sites could form a single hybrid site or the binding of L20 to a first site may inhibit binding to the second. Models of regulation compatible with our data are discussed.     

Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites

Two exceptional mechanisms of eukaryotic translation initiation have recently been identified that differ fundamentally from the canonical factor-mediated, end-dependent mechanism of ribosomal attachment to mRNA. Instead, ribosomal 40S subunits bind in a factor-independent manner to the internal ribosomal entry site (IRES) in an mRNA. These two mechanisms are exemplified by initiation on the unrelated approximately 300 nt.-long Hepatitis C virus (HCV) IRES and the approximately 200 nt.-long cricket paralysis virus (CrPV) intergenic region (IGR) IRES, respectively. Ribosomal binding involves interaction with multiple non-contiguous sites on these IRESs, and therefore also differs from the factor-independent attachment of prokaryotic ribosomes to mRNA, which involves base-pairing to the linear Shine-Dalgarno sequence. The HCV IRES binds to the solvent side of the 40S subunit, docks a domain of the IRES into the ribosomal exit (E) site and places the initiation codon in the ribosomal peptidyl (P) site. Subsequent binding of eIF3 and the eIF2-GTP/initiator tRNA complex to form a 48S complex is followed by subunit joining to form an 80S ribosome. The CrPV IRES binds to ribosomes in a very different manner, by occupying the ribosomal E and P sites in the intersubunit cavity, thereby excluding initiator tRNA. Ribosomes enter the elongation stage of translation directly, without any involvement of initiator tRNA or initiation factors, following recruitment of aminoacyl-tRNA to the ribosomal aminoacyl (A) site and translocation of it to the P site.     

Reading frame switch caused by base-pair formation between the 3'' end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli.

Watson-Crick base pairing is shown to occur between the mRNA and nucleotides near the 3' end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli. This base-pairing is similar to the mRNA-rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S-D) nucleotides of ribosome binding sites and their complements in the 1540-1535 region of 16S rRNA. mRNA-rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S-D nucleotides precisely spaced 5' on the mRNA from the frameshift site. Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S-D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing. This result implies that the 3' end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.     

The binding of protons and inositol hexakisphosphate to ligated and unligated human des-Arg141alpha-hemoglobin.

The region of Bacillus stearothermophilus strain NCA 1503 23-S ribosomal RNA protected from T1 ribonuclease digestion by the 50-S ribosomal subunit protein L1 from Escherichia coli has been established. The sequence of 115 nucleotides is compared to the analogous region in E. coli. The similar behaviour of the RNA towards the recognition of protein L1 may be explained in terms of secondary base-pairing, even though there exists almost 40% difference between the primary nucleotide sequences.     

A competition mechanism regulates the translation of the Escherichia coli operon encoding ribosomal proteins L35 and L20

Escherichia coli ribosomal protein (r-protein) L20 is essential for the assembly of the 50S ribosomal subunit and is also a translational regulator of its own rpmI-rplT operon, encoding r-proteins L35 and L20 in that order. L20 directly represses the translation of the first cistron and, through translational coupling, that of its own gene. The translational operator of the operon is 450 nt in length and includes a long-range pseudoknot interaction between two RNA sequences separated by 280 nt. L20 has the potential to bind both to this pseudoknot and to an irregular hairpin, although only one site is occupied at a time during regulation. This work shows that the rpmI-rplT operon is regulated by competition between L20 and the ribosome for binding to mRNA in vitro and in vivo. Detailed studies on the regulatory mechanisms of r-protein synthesis have only been performed on the rpsO gene, regulated by r-protein S15, and on the alpha operon, regulated by S4. Both are thought to be controlled by a trapping mechanism, whereby the 30S ribosomal subunit, the mRNA, and the initiator tRNA are blocked as a nonfunctional preternary complex. This alternative mode of regulation of the rpmI-rplT operon raises the possibility that control is kinetically and not thermodynamically limited in this case. We show that the pseudoknot, which is known to be essential for L20 binding and regulation, also enhances 30S binding to mRNA as if this structure is specifically recognised by the ribosome.     

Number of tRNA binding sites on 80 S ribosomes and their subunits

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5-20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA.     

Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA

The 16S rRNA-binding ribosomal protein S15 is a key component in the assembly of the small ribosomal subunit in bacteria. We have shown that S15 from the extreme thermophile Thermus thermophilus represses the translation of its own mRNA in vitro, by interacting with the leader segment of its mRNA. The S15 mRNA-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis. S15 binding triggers a conformational rearrangement of its mRNA into a fold that mimics the conserved three-way junction of the S15 rRNA-binding site. This conformational change masks the ribosome entry site, as demonstrated by direct competition between the ribosomal subunit and S15 for mRNA binding. A comparison of the T.thermophilus and Escherichia coli regulation systems reveals that the two regulatory mRNA targets do not share any similarity and that the mechanisms of translational inhibition are different. Our results highlight an astonishing plasticity of mRNA in its ability to adapt to evolutionary constraints, that contrasts with the extreme conservation of the rRNA-binding site.     

Interaction of the Bacillus stearothermophilus ribosomal protein S15 with its 5'-translational operator mRNA

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds both a three-helix junction in the central domain of 16 S ribosomal RNA and its cognate mRNA. Native gel mobility-shift assays show that BS15 interacts specifically and with high affinity to the 5'-untranslated region (5'-UTR) of this cognate mRNA with an apparent dissociation constant of 3(+/-0.3) nM. In order to localize the structural elements that are essential for BS15 recognition, a series of deletion mutants of the full cognate mRNA were prepared and tested in the same gel-shift assay. The minimal binding site for BS15 is a 50 nucleotide RNA showing a close secondary structure resemblance to the BS15 binding region from 16 S rRNA. There are two major structural motifs that must be maintained for high-affinity binding. The first being a purine-rich three-helix junction, and the second being an internal loop. The sequence identity of the internal loops differs greatly between the BS15 mRNA and rRNA sites, and this difference is correlated to discrimination between wild-type BS15 and a BS15(H45R) mutant. The association and dissociation kinetics measured for the 5'-UTR-BS15 interaction are quite slow, but are typical for a ribosomal protein-RNA interaction. The BS15 mRNA and 16 S rRNA binding sites share a common secondary structure yet have little sequence identity. The mRNA and rRNA may in fact present similar if not identical structural elements that confer BS15 recognition.     

Yeast ribosomal protein L25 binds to an evolutionary conserved site on yeast 26S and E. coli 23S rRNA.

The binding site of the yeast 60S ribosomal subunit protein L25 on 26S rRNA was determined by RNase protection experiments. The fragments protected by L25 originate from a distinct substructure within domain IV of the rRNA, encompassing nucleotides 1465-1632 and 1811-1861. The protected fragments are able to rebind to L25 showing that they constitute the complete protein binding site. This binding site is remarkably conserved in all 23/26/28S rRNAs sequenced to date including Escherichia coli 23S rRNA. In fact heterologous complexes between L25 and E. coli 23S rRNA could be formed and RNase protection studies on these complexes demonstrated that L25 indeed recognizes the conserved structure. Strikingly the L25 binding site on 23S rRNA is virtually identical to the previously identified binding site of E. coli ribosomal protein EL23. Therefore EL23 is likely to be the prokaryotic counterpart of L25 in spite of the limited homology displayed by the amino acid sequences of the two proteins.     

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.     

The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.     

How bacterial ribosomal protein L20 assembles with 23 S ribosomal RNA and its own messenger RNA

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.     

The structure of a ribosomal protein S8/spc operon mRNA complex

In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.     

Do eukaryotic mRNA 5' noncoding sequences base-pair with the 18 S ribosomal RNA 3' terminus?

Protein synthesis initiation on prokaryotic mRNAs involves base-pairing of a site preceding the initiation codon with the 3' terminal sequence of 16 S rRNA. It has been suggested that a similar situation may prevail in eukaryotic mRNAs. This suggestion is not based on experiments, but on observation of complementarities between mRNA 5' noncoding sequences and a conserved sequence near the 18 S rRNA 3' terminus. The hypothesis can be evaluated by comparing the number of potential binding sites found in the 5' noncoding sequences with the number of such sites expected to occur by chance. A method for computing this number is presented. The 5' noncoding sequences contain more binding sites than expected for a random RNA chain, but the same is true for 3' noncoding sequences. The effect can be traced to a clustering of purines and pyrimidines, common to noncoding sequences. In conclusion, a close inspection of the available mRNA sequences does not reveal any indication of a specific base-pairing ability between their 5' noncoding segments and the 18 S rRNA 3' terminus.     

On the structural specificity of puromycin binding to Escherichia coli ribosomes

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.     

Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

During translation, tRNAs cycle through three binding sites on the ribosome: the A, the P, and the E sites. We have determined the structures of complexes between the Haloarcula marismortui large ribosomal subunit and two different E site substrates: a deacylated tRNA acceptor stem minihelix and a CCA-acceptor end. Both of these tRNA mimics contain analogs of adenosine 76, the component responsible for a large proportion of E site binding affinity. They bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA including bases that are conserved in all three kingdoms of life. These contacts are consistent with the footprinting, protection, and cross-linking data that have identified the E site biochemically. These structures explain the specificity of the E site for deacylated tRNAs, as it is too small to accommodate any relevant aminoacyl-tRNA. The orientation of the minihelix suggests that it may mimic the P/E hybrid state. It appears that the E site on the 50S subunit was formed by only RNA in the last common ancestor of the three kingdoms, since the proteins at the E sites of H. marismortui and Deinucoccus radiodurans large subunits are not homologous.     

Saturation mutagenesis of a +1 programmed frameshift-inducing mRNA sequence derived from a yeast retrotransposon

Errors during the process of translating mRNA information into protein products occur infrequently. Frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. Despite these mechanisms, mRNA sequences have evolved that increase the frequency up to 10,000-fold. These sequences, termed programmed frameshift sites, usually consist of a heptameric nucleotide sequence, at which the change in frames occurs along with additional sequences that stimulate the efficiency of frameshifting. One such stimulatory site derived from the Ty3 retrotransposon of the yeast Saccharomyces cerevisiae (the Ty3 stimulator) comprises a 14 nucleotide sequence with partial complementarity to a Helix 18 of the 18S rRNA, a component of the ribosome's accuracy center. A model for the function of the Ty3 stimulator predicts that it base pairs with Helix 18, reducing the efficiency with which the ribosome rejects erroneous out of frame decoding. We have tested this model by making a saturating set of single-base mutations of the Ty3 stimulator. The phenotypes of these mutations are inconsistent with the Helix 18 base-pairing model. We discuss the phenotypes of these mutations in light of structural data on the path of the mRNA on the ribosome, suggesting that the true target of the Ty3 stimulator may be rRNA and ribosomal protein elements of the ribosomal entry tunnel, as well as unknown constituents of the solvent face of the 40S subunit.