Competition between erythromycin and virginiamycin for in vitro binding to the large ribosomal subunit

When the S component of virginiamycin binds in vitro to the 50 S ribosomal subunit, a change of fluorescence intensity proportional to the amount of complex formed occurs. Erythromycin competes with virginiamycin S for attachment to ribosomes, and removes previously bound virginiamycin S from its target, as revealed by spectrofluorimetric analysis. The 50 S subunits which are incubated with the M component of virginiamycin (50 S*) have an increased affinity for virginiamycin S (the association constants of virginiamycin S with ribosomes are 2.5 x 10(6) M-1 in the absence of virginiamycin M, and 15 x 10(6) M-1 in its presence). Erythromycin does not compete with virginiamycin S for attachment to 50 S* subunits nor is it able to remove virginiamycin S previously bound to the 50 S* subunit. Thus, virginiamycin M produces a change in ribosomes, which results in a tighter complex virginiamycin S-50 S* subunit. Such change does not require the presence of virginiamycin M, however, as shown by the observation that ribosomes to which labeled virginiamycin M is transiently linked bind virginiamycin S in a form that cannot be removed by erythromycin.     

Database on the structure of large ribosomal subunit RNA.

Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp.     

Database on the structure of large subunit ribosomal RNA.

The Antwerp database on large subunit ribosomal RNA now contains 607 complete or nearly complete aligned sequences. The alignment incorporates secondary structure information for each sequence. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available. Information from the database can be downloaded or searched on the rRNA WWW Server at URL http://rrna.uia.ac.be/     

Database on the structure of large ribosomal subunit RNA.

The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp.     

Database on the structure of large ribosomal subunit RNA.

The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site.     

Database on the structure of large ribosomal subunit RNA.

A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp.     

Additional Watson-Crick interactions suggest a structural core in large subunit ribosomal RNA

Two new Watson-Crick type interactions in 23S-like ribosomal RNA have been identified by comparative sequence analysis. These interactions, A1269/U2011 and C1270/G2010 (E. coli numbering) along with the previously proposed A1262/U2017 suggest an anti-parallel helical arrangement characteristic of secondary structure in the 1265/2015 region of 23S rRNA. Nested within these three interactions are three universal juxtapositions which in principle allow the formation of an irregular helix containing two additional A-G interactions and a universal A-U pair. Whether or not this extended helix is biologically significant is uncertain. The proponderance of interactions in the 1265/2015 region and its location relative to the known structural domains of 23S rRNA suggest that this region may be part of a central structural core similar to that already known in 16S rRNA.     

Evolution according to large ribosomal subunit RNA

Evolutionary trees were constructed, by distance methods, from an alignment of 225 complete large subunit (LSU) rRNA sequences, representing Eucarya, Archaea, Bacteria, plastids, and mitochondria. A comparison was made with trees based on sets of small subunit (SSU) rRNA sequences. Trees constructed on the set of 172 species and organelles for which the sequences of both molecules are known had a very similar topology, at least with respect to the divergence order of large taxa such as the eukaryotic kingdoms and the bacterial divisions. However, since there are more than ten times as many SSU as LSU rRNA sequences, it is possible to select many SSU rRNA sequence sets of equivalent size but different species composition. The topologies of these trees showed considerable differences according to the particular species set selected.The effect of the dataset and of different distance correction methods on tree topology was tested for both LSU and SSU rRNA by repetitive random sampling of a single species from each large taxon. The impact of the species set on the topology of the resulting consensus trees is much lower using LSU than using SSU rRNA. This might imply that LSU rRNA is a better molecule for studying wide-range relationships. The mitochondria behave clearly as a monophyletic group, clustering with the Proteobacteria. Gram-positive bacteria appear as two distinct groups, which are found clustered together in very few cases. Archaea behave as if monophyletic in most cases, but with a low confidence.Abbreviations LSU rRNA large subunit ribosomal RNA - SSU rRNA small subunit ribosomal RNA - JC Jukes and Cantor - JN Jin and Nei Correspondence to: R. De Wachter     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

High resolution structure of the large ribosomal subunit from a mesophilic eubacterium.

We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.     

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.     

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates.It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established.Here,we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 A resolution,revealing a half-assembled subunit.DomainsⅠ,ⅡandⅣof 25S/5.8S rRNA pack tightly into a native-like substructure,but domains Ⅲ,ⅣandⅤare not assembled.The structure contains 12 assembly factors and 19 ribosomal proteins,many of which are required for early processing of large subunit rRNA.The Brx1-Ebp2 complex would interfere with the assembly of domains Ⅳ and Ⅴ.Rpf1,Mak16,Nsa1 and Rrp1 form a cluster that consolidates the joining of domainsⅠandⅡ.Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.     

A compact three-dimensional crystal form of the large ribosomal subunit from Bacillus stearothermophilus

A new form of well-ordered three-dimensional crystals of intact 50 S ribosomal subunits from Bacillus stearothermophilus have been obtained. Electron micrographs of positively stained sections of these crystals revealed that the ribosomal particles are packed closely. The cell parameters have been determined. Representative electron micrographs and their computed contoured filtered images are shown.     

RNA:RNA interactions in the large subunit ribosomal RNA of Euglena gracilis

In Euglena gracilis, the cytoplasmic large subunit (LSU) rRNA is composed of 14 discrete small RNA species that must somehow interact in the functional ribosome. We have isolated native complexes of Euglena rRNA and show here that the largest of these complexes contains eight of the 14 LSU rRNA species. Several of these small rRNA species are able to associate in vitro to reform an isolated domain of LSU rRNA structure.     

The structures of four macrolide antibiotics bound to the large ribosomal subunit

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.     

Database on the structure of small ribosomal subunit RNA.

The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library.