Evidence against an Interaction between the mRNA downstream box and 16S rRNA in translation initiation.

Based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mRNAs to bases 1469 to 1483 in helix 44 of 16S rRNA (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the Shine-Dalgarno (SD)/anti-Shine-Dalgarno (aSD) interaction. Computer modeling of helix 44 on the 30S subunit shows that the topography of the 30S ribosome does not allow a simultaneous db-adb interaction and placement of the initiation codon in the ribosomal P site. Thus, the db-adb interaction cannot substitute for the SD-aSD interaction in translation initiation. We have always argued that any contribution of the db-adb interaction should be most apparent on mRNAs devoid of an SD sequence. Here, we show that 30S ribosomes do not bind to leaderless mRNA in the absence of initiator tRNA, even when the initial coding region shows a 15-nucleotide complementarity (optimal fit) with the putative adb. In addition, an optimized db did not affect the translational efficiency of a leaderless lambda cI-lacZ reporter construct. Thus, the db-adb interaction can hardly serve as an initial recruitment signal for ribosomes. Moreover, we show that different leaderless mRNAs are translated in heterologous systems although the sequence of the putative adb's within helix 44 of the 30S subunits of the corresponding bacteria differ largely. Taken our data together with those of others (M. O'Connor, T. Asai, C. L. Squires, and A. E. Dahlberg, Proc. Natl. Acad. Sci. USA 96:8973-8978, 1999; A. La Teana, A. Brandi, M. O'Connor, S. Freddi, and C. L. Pon, RNA 6:1393-1402, 2000), we conclude that the db does not base pair with the adb.

Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui.

Insertions were introduced by a two-step mutagenesis procedure into each of five double-helical regions of Escherichia coli 23 S rRNA, so as to extend the helix concerned by 17 bp. The helices chosen were at sites within the 23 S molecule (h9, h25, h45, h63 and h98) where significant length variations between different species are known to occur. At each of these positions, with the exception of h45, there are also significant differences between the 23 S rRNAs of E. coli and Haloarcula marismortui. Plasmids carrying the insertions were introduced into an E. coli strain lacking all seven rrn operons. In four of the five cases the cells were viable and 50 S subunits could be isolated; only the insertion in h63 was lethal. The modified subunits were examined by cryo-electron microscopy (cryo-EM), with a view to locating extra electron density corresponding to the insertion elements. The results were compared both with the recently determined atomic structure of H. marismortui 23 S rRNA in the 50 S subunit, and with previous 23 S rRNA modelling studies based on cryo-EM reconstructions of E. coli ribosomes. The insertion element in h45 was located by cryo-EM at a position corresponding precisely to that of the equivalent helix in H. marismortui. The insertion in h98 (which is entirely absent in H. marismortui) was similarly located at a position corresponding precisely to that predicted from the E. coli modelling studies. In the region of h9, the difference between the E. coli and H. marismortui secondary structures is ambiguous, and the extra electron density corresponding to the insertion was seen at a location intermediate between the position of the nearest helix in the atomic structure and that in the modelled structure. In the case of h25 (which is about 50 nucleotides longer in H. marismortui), no clear extra cryo-EM density corresponding to the insertion could be observed.

A protonated base pair participating in rRNA tertiary structural interactions.

In the recently published X-ray crystallographic structure for the 50S subunit of Haloarcula marismortui ribosomes, residue U2546 of the 23S rRNA forms a non-Watson-Crick base pair with U2610. The corresponding residues in the secondary structure of the Escherichia coli 23S molecule are U2511 and C2575, and it follows that the latter base (C2575) should be protonated in order to form a base pair that is isostructural with its counterpart in H.marismortui. This prediction was demonstrated experimentally by reduction with sodium borohydride followed by primer extension analysis; borohydride is able to reduce positively charged bases, yielding products which block reverse transcription. In the course of the analysis a further charged base pair (AH(+)1528-G1543) was identified in the E.coli 23S molecule. Both charged pairs (U2511-CH(+)2575 and AH(+)1528-G1543) were only observed in the context of the intact ribosomal subunit and were not seen in deproteinized rRNA.

The bacterial ribosome at atomic resolution.

X-ray crystallographic structures have just been published for the 30S ribosomal subunit of Thermus thermophilus at 3.4 A resolution and for the 50S subunit of Haloarcula marismortui at 2.4 A. These eagerly awaited structures will provide an enormous boost to research into the mechanisms involved in protein biosynthesis.

The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5 A resolution.

The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an unfiltered version of the recently published 50 S reconstruction at 7.5 A resolution. At this resolution, the EM density shows a well-defined network of fine structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A or 20 A. Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit. The post-transcriptionally modified bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure.

Bacterial ribosome structure: approaching atomic resolution.

Progress in the X-ray crystallography of bacterial ribosomes or their subunits has become so rapid that we are now seeing the publication of near-complete atomic structures for both the 16S and 23S rRNA molecules from thermophilic and halophilic ribosomes. At the same time, cryo-electron microscopic studies on Escherichia coli ribosomes in various functional states are now yielding structures at single figure resolution and still improving. Combined with all of the available biochemical information, these new results will generate a quantum leap in our understanding of the structures and mechanisms involved in protein biosynthesis.

The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule.

Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple cross-links from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.

The Database of Ribosomal Cross-links: an update.

The Database of Ribosomal Cross-links (DRC) was created in 1997. Here we describe new data incorporated into this database and several new features of the DRC. The DRC is freely available via World Wide Web at http://visitweb.com/database/ or http://www. mpimg-berlin-dahlem.mpg.de/ approximately ag_ribo/ag_brimacombe/drc/

The Escherichia coli large ribosomal subunit at 7.5 A resolution.

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.

Flexibility of the nascent polypeptide chain within the ribosome--contacts from the peptide N-terminus to a specific region of the 30S subunit.

The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887-895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked. The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit. The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the 'ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA.

Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution.

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome.

23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118. In each case, the 3' terminus of the 5' fragment was ligated to radioactively labeled 4-thiouridine 5'-,3'-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits. The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes. Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed. The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510. In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993. The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome. This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.

The environment of 5S rRNA in the ribosome: cross-links to the GTPase-associated area of 23S rRNA.

Two photoreactive diazirine derivatives of uridine were used to study contacts between 5S rRNA and 23 rRNA in situ in Escherichia coli ribosomes. 2'-Amino-2'-deoxy-uridine or 5-methyleneaminouridine were introduced into 5S rRNA by T7 transcription. After incorporation of these uridine analogues into the transcript their amino groups were modified with 4-[3-(trifluoromethyl)-3 H -diazirin-3-yl]benzyl isothiocyanate or the N -hydroxysuccinimide ester of 4-[3-(trifluoromethyl)-3 H -diazirin-3-yl]benzoic acid respectively. 5S rRNA carrying the photoreactive diazirine groups (referred to as the 2'-aminoribose derivative and the 5-methyleneamino derivative respectively) was reconstituted into 50S subunits or 70S ribosomes. After mild UV irradiation cross-links formed to 23S rRNA were analysed by standard procedures. All of the observed cross-links involved residue U89 of the 5S rRNA. Three nucleotides of 23S rRNA were cross-linked to this residue with the 5-methyleneamino derivative, namely U958, G1022 and G1138. With the 2'-aminoribose derivative a single cross-link was found, to U958. The significance of these cross-links for our understanding of the structure and function of 5S rRNA and its environment in the ribosome are discussed.

Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA.

Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.

The path of the growing peptide chain through the 23S rRNA in the 50S ribosomal subunit; a comparative cross-linking study with three different peptide families.

As part of a programme to investigate the path of the nascent peptide through the large ribosomal subunit, peptides of different lengths (up to 30 amino acids), corresponding to the signal peptide sequence and N-terminal region of the Escherichia coli ompA protein, were synthesized in situ on E.coli ribosomes. The peptides each carried a diazirine moiety attached to their N-terminus which, after peptide synthesis, was photoactivated so as to induce cross-links to the 23S rRNA. The results showed that, with increasing length, the peptides became progressively cross-linked to sites in Domains V, II, III and I of the 23S rRNA, in a similar manner to that previously observed with a family of peptides derived from the tetracycline resistance gene. However, the cross-links to Domain III appeared at a shorter peptide length (12 aa) in the case of the ompA sequence, and an additional cross-link in Domain II (localized to nt 780-835) was also observed from this peptide. As with the tetracycline resistance sequence, peptides of all lengths were still able to form cross-links from their N-termini to the peptidyl transferase centre in Domain V. A further set of peptides (30 or 50 aa long), derived from mutants of the bacteriophage T4 gene 60 sequence, did not show the cross-links to Domain III, but their N-termini were nevertheless cross-linked to Domain I and to the sites in Domains II and V. The ability of relatively long peptides to fold back towards the peptidyl transferase centre thus appears to be a general phenomenon.

The Database of Ribosomal Cross links (DRC).

The Database of Ribosomal Cross-links (DRC) provides a complete collection of all the published data produced by cross-linking studies on the Escherichia coli ribosome, as well as on its components and functional ligands. The DRC currently includes data on 986 cross-links from >100 research papers, yielded by >40 different reagents. For each cross-link, information is given concerning its location in the ribosome, the chemical or photochemical reagent applied, a brief description of the method(s) used to locate the cross-link, and the literature reference. The DRC is freely available via the World Wide Web at: http://Ribosome.Genebee.MSU.SU/DRC/ or at http://WWW:MPIMG-Berlin-Dahlem.MPG.DE/[symbol: see text]baranov/DRC/

Visualization of elongation factor Tu on the Escherichia coli ribosome.

The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre. Domain 1 (the G domain) of the EF-Tu is bound both to the L7/L12 stalk and to the 50S body underneath the stalk, whereas domain 2 is oriented towards the S12 region on the 30S subunit.

Decoding the translational termination signal: the polypeptide chain release factor in Escherichia coli crosslinks to the base following the stop codon.

Protein release factors act like tRNA analogues in decoding translational stop signals. Statistical analysis of the sequences at translational stop sites and functional studies with particular signals indicate this mimicry involves an increase in the length of the signal in the mRNA. The base following the stop codon (+4 base) is of particular interest because it has a strong influence on the competitiveness of the stop signal at recoding sites, suggesting it might form part of the release factor recognition element. Site-directed crosslinking from the +4 base showed that it is in close proximity to the Escherichia coli release factor-2 in a termination complex, a prerequisite for the +4 base being part of the recognition element. Fingerprinting analysis indicates that crosslinking to the release factor occurred from both +1 and +4 positions of the stop signal in the same RNA molecule. This provides more evidence that the +4 base may be an integral part of the decoding signature in the mRNA during the termination phase of protein biosynthesis.