Positioning of mRNA codons with respect to 18S rRNA at the P and E sites of human ribosome

Positioning of each nucleotide of the E site and the P site bound codons with respect to the 18S rRNA on the human ribosome was studied by cross-linking with mRNA analogs, derivatives of the hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group on the second or the third uracil, and a derivative of the dodecaribonucleotide UUAGUAUUUAUU with a similar group on the guanine residue. The location of the modified nucleotides at any mRNA position from -3 to +3 (position +1 corresponds to the 5' nucleotide of the P site bound codon) was adjusted by the cognate tRNAs. A modified uridine at positions from -1 to +3 cross-linked to nucleotide G1207 of the 18S rRNA, and to nucleotide G961 when it was in position -2. A modified guanosine cross-linked to nucleotide G1207 if it was in position -3 of the mRNA. These data indicate that nucleotide G961 of the 18S rRNA is close only to mRNA positions -3 and -2, while G1207 is in the vicinity of positions from -3 to +3. The latter suggests that there is a sharp turn between the P and E site bound codons that brings nucleotide G1207 of the 18S rRNA close to each nucleotide of these codons. This correlates well with X-ray crystallographic data on bacterial ribosomes, indicating existence of a sharp turn between the P site and E site bound codons near a conserved nucleotide G926 of the 16S rRNA (corresponding to G1207 in 18S rRNA) close to helix 23b containing the conserved nucleotide 693 of the 16S rRNA (corresponding exactly to G961 of the 18S rRNA).     

The sequence of the nucleotides at the alpha-sarcin cleavage site in rat 28 S ribosomal ribonucleic acid

The sequence of the 521 nucleotides at the 3' end of a rat 28 S rRNA gene was determined. The region encompasses the site of cleavage of 28 S rRNA by the cytotoxin alpha-sarcin. The toxin hydrolyzes a phosphodiester bond on the 3' side of a guanine residue 393 nucleotides from the 3' end. The alpha-sarcin domain is composed of a purine-rich sequence of 14 highly conserved nucleotides.     

A story: unpaired adenosine bases in ribosomal RNAs

In 1985 an analysis of the Escherichia coli 16 S rRNA covariation-based structure model revealed a strong bias for unpaired adenosines. The same analysis revealed that the majority of the G, C, and U bases were paired. These biases are (now) consistent with the high percentage of unpaired adenosine nucleotides in several structure motifs.An analysis of a larger set of bacterial comparative 16 S and 23 S rRNA structure models has substantiated this initial finding and revealed new biases in the distribution of adenosine nucleotides in loop regions. The majority of the adenosine nucleotides are unpaired, while the majority of the G, C, and U bases are paired in the covariation-based structure model. The unpaired adenosine nucleotides predominate in the middle and at the 3' end of loops, and are the second most frequent nucleotide type at the 5' end of loops (G is the most common nucleotide). There are additional biases for unpaired adenosine nucleotides at the 3' end of loops and adjacent to a G at the 5' end of the helix. The most prevalent consecutive nucleotides are GG, GA, AG, and AA. A total of 70 % of the GG sequences are within helices, while more than 70 % of the AA sequences are unpaired. Nearly 50 % of the GA sequences are unpaired, and approximately one-third of the AG sequences are within helices while another third are at the 3' loop.5' helix junction. Unpaired positions with an adenosine nucleotide in more than 50 % of the sequences at the 3' end of 16 S and 23 S rRNA loops were identified and arranged into the A-motif categories XAZ, AAZ, XAG, AAG, and AAG:U, where G or Z is paired, G:U is a base-pair, and X is not an A and Z is not a G in more than 50 % of the sequences. These sequence motifs were associated with several structural motifs, such as adenosine platforms, E and E-like loops, A:A and A:G pairings at the end of helices, G:A tandem base-pairs, GNRA tetraloop hairpins, and U-turns.     

Comparative Surface Structure of 16S Ribosomal Ribonucleic Acid of 30S Ribosomes of Procaryotic Cells

Ribonuclease T(1) treatment of 30S ribosomes of Escherichia coli converts a large region at the 3' OH end of 16S ribosomal ribonucleic acid (rRNA) to low-molecular-weight RNA. The final 25 nucleotides at the 3' terminus of the molecule emerge relatively intact, whereas most of the region "upstream," for about 150 nucleotides, is converted to oligonucleotides. Identical enzyme treatment generates a fragment of about 60 nucleotides from the middle of 16S rRNA (section D'). To determine whether there are similar sequences in other bacteria, which occupy similar accessible surface locations, we treated 30S ribosomes from Azotobacter vinelandii and Bacillus stearothermophilus with RNase T(1). In each case, a fragment of RNA about 25 nucleotides in length containing the 3' OH end of 16S rRNA and a fragment of about 60 nucleotides in length similar, but not identical, in oligonucleotide composition to section D' of E. coli 16S rRNA were obtained from nuclease-treated 30S ribosomes. These data indicate that, although the primary structure at the 3' end and the middle (section D') of the various 16S rRNA's is not completely conserved, their respective conformations are conserved. A number of identical oligonucleotides were found in the low-molecular-weight fraction obtained from RNase T(1)-treated E. coli, A. vinelandii, and B. stearothermophilus 30S ribosomes. These results show that identical RNase T(1)-sensitive sequences are present in all three bacteria. Hydrolysis of these regions leads to the production of the fragments 25 and 60 nucleotides in length.     

The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts

The chloroplast genes coding for the 16S ribosomal RNA from several spectinomycin-resistant Nicotiana mutants were analyzed. Two classes of mutants were identified. In one class, a G to A base transition is found at position 1140 of the tobacco-chloroplast 16S rRNA gene, which eliminates an AatII restriction endonuclease site. This base transition is proximal to a mutation previously described for spectinomycin resistance in Escherichia coli. In the other class, a novel G to A transition is found at position 1012 of the 16S rRNA gene. Although the mutations in the two classes are 128 nucleotides apart, the secondary structure model for 16S rRNA suggests that the two mutated nucleotides are in spatial proximity on opposite sides of a conserved stem structure in the 3' region of the molecule. Phylogenetic evidence is presented linking this conserved stem with spectinomycin resistance in chloroplasts. Perturbation of the stem is proposed to be the molecular-genetic basis for rRNA-dependent spectinomycin resistance.     

The structure of helix III in Xenopus oocyte 5 S rRNA: an RNA stem containing a two-nucleotide bulge

The solution structure of an oligonucleotide containing the helix III sequence from Xenopus oocyte 5 S rRNA has been determined by NMR spectroscopy. Helix III includes two unpaired adenosine residues, flanked on either side by G:C base-pairs, that are required for binding of ribosomal protein L5. The consensus conformation of helix III in the context provided by this oligonucleotide has the two adenosine residues located in the minor groove and stacked upon the 3' flanking guanosine residue, consistent with biochemical studies of free 5 S rRNA in solution. A distinct break in stacking that occurs between the first adenosine residue of the bulge and the flanking 5' guanosine residue exposes the base of the adenosine residue in the minor groove and the base of the guanosine residue in the major groove. The major groove of the helix is widened at the site of the unpaired nucleotides and the helix is substantially bent; nonetheless, the G:C base-pairs flanking the bulge are intact. The data indicate that there may be conformational heterogeneity centered in the bulge region. The corresponding adenosine residues in the Haloarcula marismortui 50 S ribosomal subunit form a dinucleotide platform, which is quite different from the motif seen in solution. Thus, the conformation of helix III probably changes when 5 S rRNA is incorporated into the ribosome.     

Is there a special function for U.G basepairs in ribosomal RNA?

U.G basepairs are well-established elements of RNA structure. The geometry of this pair is different, however, from classical Watson-Crick basepairs. This leads to an unusual stacking of the basepair: overlap with the basepair at the 5' side of the U (and the 3' side of the G) is strong (stacked) while it is weak with the basepair on the other side (destacked). The closure of an RNA helix by a U.G pair will be energetically unfavourable when the U residue occupies the 5' end. In transfer RNA there is a strong selection against a 'destacked' U.G pair at helix ends. In the 16S rRNA model of Escherichia coli there are 72 U.G pairs of which 36 or 22 occupy a helix end, depending on how such an end is defined. There is a slight preference for 'stacked' U.G's in these positions. It is remarkable, however, that of 13 very conserved U.G pairs in the 16S (-like) rRNA, 7 occur at helix ends and that 5 of these have the 'destacked' configuration. It is suggested that these pairs, if they exist at all in a hydrogen-bounded form, are stabilized by co-axial stacking with other helices or by interaction with a protein.     

Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: a crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5'-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions -3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position -3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.     

Escherichia coli initiation factor 3 protein binding to 30S ribosomal subunits alters the accessibility of nucleotides within the conserved central region of 16S rRNA

Translational initiation factor 3 (IF3) is an RNA helix destabilizing protein which interacts with strongly conserved sequences in 16S rRNA, one at the 3' terminus and one in the central domain. It was therefore of interest to identify particular residues whose exposure changes upon IF3 binding. Chemical and enzymatic probing of central domain nucleotides of 16S rRNA in 30S ribosomal subunits was carried out in the presence and absence of IF3. Bases were probed with dimethyl sulfate (DMS), at A(N-1), C(N-3), and G(N-7), and with N-cyclohexyl-N'-[2-(N-methyl-4-morpholinio)ethyl] carbodiimide p-toluenesulfonate (CMCT), at G(N-1) and U(N-3). RNase T1 and nuclease S1 were used to probe unpaired nucleotides, and RNase V1 was used to monitor base-paired or stacked nucleotides. 30S subunits in physiological buffers were probed in the presence and absence of IF3. The sites of cleavage and modification were detected by primer extension. IF3 binding to 30S subunits was found to reduce the chemical reactivity and enzymatic accessibility of some sites and to enhance attack at other sites in the conserved central domain of 16S rRNA, residues 690-850. IF3 decreased CMCT attack at U701 and U793 and V1 attack at G722, G737, and C764; IF3 enhanced DMS attack at A814 and V1 attack at U697, G833, G847, and G849. Many of these central domain sites are strongly conserved and with the conserved 3'-terminal site define a binding domain for IF3 which correlates with a predicted cleft in two independent models of the 30S ribosomal subunit.     

Mouse rDNA: sequences and evolutionary analysis of spacer and mature RNA regions.

Two regions of mouse rDNA were sequenced. One contained the last 323 nucleotides of the external transcribed spacer and the first 595 nucleotides of 18S rRNA; the other spanned the entire internal transcribed spacer and included the 3' end of 18S rRNA, 5.8S rRNA, and the 5' end of 28S rRNA. The mature rRNA sequences are very highly conserved from yeast to mouse (unit evolutionary period, the time required for a 1% divergence of sequence, was 30 X 10(6) to 100 X 10(6) years). In 18S rRNA, at least some of the evolutionary expansion and increase in G + C content is due to a progressive accretion of discrete G + C-rich insertions. Spacer sequence comparisons between mouse and rat rRNA reveal much more extensive and frequent insertions and substitutions of G + C-rich segments. As a result, spacers conserve overall G + C richness but not sequence (UEP, 0.3 X 10(6) years) or specific base-paired stems. Although no stems analogous to those bracketing 16S and 23S rRNA in Escherichia coli pre-rRNA are evident, certain features of the spacer regions flanking eucaryotic mature rRNAs are conserved and could be involved in rRNA processing or ribosome formation. These conserved regions include some short homologous sequence patterns and closely spaced direct repeats.     

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 Shine and Dalgarno hypothesis for termination: the 3' terminus of the 16S rRNA of the Escherichia coli ribosome can be modified or base-paired with a complementary oligonucleotide without affecting termination in vitro

The occurrence of the nucleotides "...CCUUAOH" at the 3' terminus of the 16S rRNA of the small subunit of the Escherichia coli ribosome led to the suggestion that they may have a direct base pairing with the termination codon in the termination event of protein biosynthesis (Shine and Dalgarno 1974). We have examined this concept with two approaches, firstly using a 30S subunit whose 16S rRNA has been modified with a fluorescein moiety on the terminal adenosine together with the antibody against the moiety, and secondly with an oligonucleotide, UAAGG, complementary to the terminal pentanucleotide sequence of the rRNA. Collectively the data suggest that the nucleotides at the 3' terminus of 16S rRNA are not critically involved in base pairing during termination codon recognition.     

Action of Ribonuclease T1 on 30S Ribosomes of Escherichia coli and Its Role in Sequence Studies on 16S Ribonucleic Acid

Two large ribonucleic acid (RNA) fragments have been obtained from T1-RNase-treated 30S ribosomes of Escherichia coli. One fragment, about 475 nucleotides long, contains all the unique oligonucleotides found by Fellner and associates in sections of 16S RNA designated P, E, E', and K, and one-half the large oligonucleotides of section A. The other large fragment is about 300 nucleotides long and contains the oligonucleotides found in sections C, C', C'. The isolation of these large fragments seems to confirm the arrangement of sections within 16S RNA. There are also recovered from nuclease-treated ribosomes three small fragments, one (120 nucleotides long) from the 5' end, one (26 nucleotides long) from the 3' OH end of the chain, and another section (66 nucleotides long) from the middle of the 16S RNA chain. Small molecular weight material is also generated by nuclease treatment, and about half this material is derived from a region close to the 3' OH end of the 16S RNA chain. This indicates that the most accessible part of the rRNA of E. coli 30S ribosomes is a region 100 to 150 nucleotides long near the 3' end of the chain. A general scheme is proposed to explain the generation of the various-sized RNA products from the rRNA of the 30S ribosome.     

Escherichia coli 16S rRNA 3''-end formation requires a distal transfer RNA sequence at a proper distance.

The 16S rRNA species in bacterial precursor rRNAs is followed by two evolutionarily conserved features: (i) a double-stranded stem formed by complementary sequences adjacent to the 5' and 3' ends of the 16S rRNA; and (ii) a 3'-transfer RNA sequence. To assess the possible role of these features, plasmid constructs with precursor-specific features deleted were tested for their capacity to form mature rRNA. Stem-forming sequences were dispensable for both 5' and 3' terminus formation; whereas an intact spacer tRNA positioned greater than 24 nucleotides downstream of the 16S RNA sequence was required for correct 3'-end maturation. These results suggest that spacer tRNA at an appropriate location helps form a conformation obligate for pre-rRNA processing, perhaps by binding to a nascent binding site in preribosomes. Thus, spacer tRNAs may be an obligate participant in ribosome formation.