Dealing with stable structures at ribosome binding sites: bacterial translation and ribosome standby.

Bacterial ribosomes have great difficulties to initiate translation on stable structures within mRNAs. Translational coupling and induced structure changes are strategies to open up inhibitory RNA structures encompassing ribosome binding sites (RBS). There are, however, mRNAs in which stable structures are not unfolded, but that are nevertheless efficiently initiated at high rates. de Smit and van Duin(1) proposed a "ribosome standby" model to theoretically solve this paradox: the 30S ribosome binds nonspecifically to an accessible site on the mRNA (standby site), waiting for a transient opening of a stable RBS hairpin. Upon unfolding, the 30S subunit relocates to form a productive initiation complex. Recent reports have provided experimental support for this model. This review will describe and compare two different flavors of standby sites, their properties, and their likely implications. We also discuss the possibility that ribosome standby may be a more general strategy to obtain high translation rates.     

The location of the first AUG codons in cowpea mosaic virus RNAs.

We have made use of the known sequence of the 5' ends of both CPMV RNAs to synthesise an oligodeoxynucleotide which can prime second-strand DNA synthesis on full-length cDNA copies of both RNAs. By priming synthesis in the presence of dideoxynucleoside triphosphates, we have determined the positions of the first AUG codons in each RNA. These occur at positions 115 and 207 on M and B RNA respectively. By using a cloned double-stranded DNA fragment derived from near the 5' end of M RNA as a primer additional sequence from the 5' terminal region of M RNA has been obtained.     

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).     

Isolation of specific ribosome binding sites from single-stranded DNA.

The ability of Escherichia coli ribosomes to protect small specific regions of single-stranded bacteriophage DNA from digestion by pancreatic DNAase has been investigated. A procedure is described by which ribosome-protected fragments can be isolated from the DNA of bacteriophage f1 and φX174. Size determination by polyacrylamide gel electrophoresis or thin layer homochromatography together with fingerprinting analysis following chemical depurination or digestion with E. coli endonuclease IV were employed to show that these fragments represent a small specific portion of these DNAs. The protection reaction is largely dependent upon components necessary for ribosome binding to mRNA, including GTP, formylmethionyl-tRNA, and initiation factors. Thus, ribosomal binding to DNA mimics the ribosome-mRNA interaction. Furthermore, the regions in f1 and φX174 DNA which are protected differ in sequence from each other.When E. coli endonuclease IV is substituted for pancreatic DNAase in the ribosome protection reaction, a fragment of φX174 DNA is obtained about 150 bases in length which contains all of the pyrimidine tracts in the shorter 50-base fragment obtained with pancreatic DNAase, and a number of additional polypyrimidines.Double-stranded DNAs such as φX174 replicative form do not bind at all to ribosomes in their native state. Heat denaturation of such double-stranded DNAs allows ribosome binding. Protection of the same specific regions as those protected in single-stranded φX174 DNA was observed. A similar specific protection was observed following heat denaturation and ribosome binding with DNA from polyoma virus.     

Sequence and symmetry in ribosome binding sites of bacteriophage f1 RNA

RNA was synthesized in vitro from α-³²P-labeled ribonucleoside triphosphates with Escherichia coli RNA polymerase from covalently closed, circular, double-stranded DNA isolated from cells infected with bacteriophage f1. This RNA, which serves as an efficient message in vitro, was bound to ribosomes and the initiation complexes were digested with bovine pancreatic ribonuclease. Ribosome-protected fragments were isolated, purified and separated by two-dimensional analysis using electrophoresis and homochromatography. Sequence analysis, taking advantage of the ability to determine nearest neighbors, was done by conventional techniques.The sequences of the ribosome-protected fragments were found to fall into three classes. One sequence corresponds to the amino-terminal region of the protein product of f1 gene V, a DNA binding protein. It is proposed that a second sequence may correspond to the amino-terminal region of a precursor to the major coat protein. No assignment has yet been made for the third sequence.Comparisons are made between these three sequences and others that are available, both in terms of sequence features that have been pointed out earlier, and in terms of certain considerations of symmetry and syntax² prominent in these binding site sequences that have not been discussed before.     

Two ribosome binding sites from the gene 0.3 messenger RNA of bacteriophage T7

Ribosome-protected regions have been isolated and analyzed from the bacteriophage T7 gene 0.3 mRNA labeled in vivo. Two discrete sites which are nearly equally protected by ribosomes are obtained from what was previously assumed to be a monocistronic message. Use of appropriate T7 deletion mutant RNAs has allowed mapping of both ribosome-recognized regions. Site a is positioned very close to the 5′ terminus of the mRNA and is apparently the initiator region for the major gene 0.3 protein, which acts to overcome the host DNA restriction system. Site b is located within several hundred nucleotides of the 3′ end of the RNA and probably initiates synthesis of a small polypeptide of unknown function. Both ribosome binding sites exhibit features common to other initiator regions from Escherichia coli and bacteriophage mRNAs. The proximity of site a to the RNase III cleavage site at the left end of gene 0.3 may explain why processing by RNase III is required for efficient translation of the major gene 0.3 protein.     

Identification of features in 5' terminal fragments from reovirus mRNA which are important for ribosome binding

Four types of experiments were carried out with reovirus messenger RNAs or with 5′ terminal fragments of known sequence to identify features in mRNA which appear to be important for formation of initiation complexes with ribosomes. With a number of reovirus mRNAs, 40S initiation complexes had been previously shown to protect a significantly larger segment of the RNA (including the 5′ terminal m7G) than that protected by 80S initiation complexes. Each 80S-protected sequence had an AUG codon and was a subset of the 40S-protected sequence from the same message. When 40S- and 80S-protected fragments were tested for ability to rebind to ribosomes, the 80S-protected fragments showed considerably lower binding ability, implying that the “extra” sequences protected by 40S initiation complexes contribute to ribosome attachment. Nevertheless, wheat germ ribosomes select the same 5′ terminal initiation site in each reovirus mRNA, irrespective of the presence or absence of m7G on the message. This was demonstrated by comparing fingerprints of the ribosome-protected regions obtained with methylated versus unmethylated RNA. The contribution of m7G to formation of initiation complexes is therefore quantitative rather than qualitative. Limited T1 RNAase digestion of isolated 5′ terminal fragments from several reovirus messages generated a series of smaller fragments which were analyzed for ability to rebind to ribosomes. Partial digestion products up to 30 nucleotides in length which retained the 5′ cap but not the AUG codon were unable to associate stably with ribosomes, whereas every AUG-containing fragment that was analyzed was able to form initiation complexes. The efficiency of binding of certain AUG-containing fragments, however, was reduced by removal of either the 5′ terminal region, including the cap, or of sequences comprising the beginning of the coding region, on the 3′ side of the AUG. Complex formation between messenger RNA and ribosomes was inhibited by the trinucleotide AUG, but not by various other oligonucleotides. Although the inhibition was specific, a vast excess of trinucleotide was required for moderate inhibition of 80S complex formation, and the same concentration of AUG failed to inhibit formation of 40S initiation complexes.     

Initiation of translation at AUC, AUA and AUU codons in Escherichia coli

A truncated form of the HBL murein hydrolase, encoded by the temperate bacteriophage HB-3, was cloned in a pUC-derivative and translated in Escherichia coli using AUC as start codon, as confirmed by biochemical, immunological, and N-terminal analyses. Using site-directed mutagenesis, we have changed this AUC codon into AUA, AUU and AUG codons. The relative translation efficiencies for these triplets were about 5% for AUC and AUU and 7.5% for AUA compared to that of AUG codon. In the same gene arrangement E. coli beta-galactosidase was also translated at moderate efficiency using AUC as initiator.     

Initiation of protein synthesis from the A site of the ribosome

Positioning of the translation initiation complex on mRNAs requires interaction between the anticodon of initiator Met-tRNA, associated with eIF2-GTP and 40S ribosomal subunit, and the cognate start codon of the mRNA. We show that an internal ribosome entry site located in the genome of cricket paralysis virus can form 80S ribosomes without initiator Met-tRNA, eIF2, or GTP hydrolysis, with a CCU triplet in the ribosomal P site and a GCU triplet in the A site. P-site mutagenesis revealed that the P site was not decoded, and protein sequence analysis showed that translation initiates at the triplet in the A site. Translational initiation from the A site of the ribosome suggests that the repertoire of translated open reading frames in eukaryotic mRNAs may be greater than anticipated.     

Leaky scanning and scanning-independent ribosome migration on the tricistronic S1 mRNA of avian reovirus

The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3'-proximal ORF encoding the sigmaC protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5'-end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5'-proximal ORFs, however, have little to no effect on translation of the 3'-proximal sigmaC ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5'-truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated sigmaC translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning- and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that sigmaC translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of sigmaC translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.     

Interaction between the erythromycin and chloramphenicol binding sites on the Escherichica coli ribosome.

The effects of chloramphenical on the binding kinetics of a fluorescein isothiocyanate derivative of 9(S)-erythromycylamine with 70S and 50S ribosomes have been studied by direct fluorimetric measurements. While chloramphenicol had little effect on the second-order 70S binding rate of the erythromycin analogue, it substantially reduced the dissociation rate of the fluorescent antibiotic-70S ribosome complex. This could be explained by simultaneous binding of both antibiotics to the 70S ribosome. The kinetic results suggest that chloramphenicol-saturated 70S particles bind the erythromycin analogue four times stronger and this was confirmed by direct binding studies. In additon, chloramphenicol causes a twofold increase in the intrinsic fluorescence of the 70S-bound analogue. This increase in fluorescence was used to study the kinetics of chloramphenicol binding to 70S ribosomes containing the fluorescent derivative. The fluorescence change followed first-order kinetics, suggesting that chloramphenicol induces a conformational change in the 70S particle. This could explain both its effect on erythromycin binding and on the fluorescence of bound analogue. Less detailed results with the 50S particle indicate a qualitively similar picture of erythromycin-chloramphenicol interactions.     

Location of ribosome binding sites on the right end of lambda DNA.

The locations of ribosome binding sites on the right end of bacteriophage lambda DNA were determined by measurement of the positions of ribosomes bound to single-stranded DNA visualized by electron microscopy. A total of ten ribosome binding sites were found between map co-ordinates 0.90 and 1.0. Four of these ribosome binding sites probably correspond to the polypeptide initiation sites for genes Q (0.910), S (0.928), R (0.936) and Rz (0.945). Six other ribosome binding sites were found which presumably indicate the presence of new lambda genes. Four of these ribosome binding sites (0.958, 0.967, 0.975, 0.995) are located to the right of Rz, which is the most rightward known lambda gene. A ribosome binding site is located at 0.923, between genes Q and S, in or near the 6 S RNA sequence. Another is located left of gene Q at 0.900.     

Effects of GUG and AUG initiation codons on the expression of lacZ in Escherichia coli

We have replaced the ribosomal binding site (RBS) of the lacZ gene of E. coli by those of the maturation (A) gene of phage MS2 and that of the tufA gene. Both RBSs contain a GUG initiation codon. The expression with the tufA RBS is at least 25-fold higher than with the phage RBS. Changing the GUG into AUG results in a 3-fold increase in expression in both cases. In general, higher expression is accompanied by an increase of lac-specific mRNA. It is argued that this is a consequence of the more efficient translation of the mRNA.     

Interaction of tRNAs with the ribosome at the A and P sites.

In vitro transcribed tRNA(Phe) analogues from Escherichia coli containing up to four randomly distributed A, G, U or C phosphorothioated nucleotides were used to investigate contact patterns with the ribosome in the A and P sites. The tRNAs were biologically active. Molecular iodine (I2) can trigger a break in the sugar-phosphate backbone at phosphorothioated positions of the ribosomal bound tRNAs if contacts with ribosomal components do not prevent access of the iodine. Highly differentiated protection patterns were found which were strikingly different in the A and P sites, respectively. Strong protections accumulated in the T psi C loop and no protection was seen in the extra-arm region in both sites, whereas the phosphates in the anticodon loop are more strongly protected in the A site. Strong common protections in both the A and P sites were found neighbouring universally or semi-universally conserved bases in prominent regions of the tertiary structure of tRNAs: Y11, Y32, U33, psi55, C56, A58 and Y60. These bases are therefore candidates for 'identity elements' in ribosomal tRNA recognition. The data further indicate that tRNAs change their conformations upon binding to either ribosomal site.     

mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-Δ disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring β-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position ?3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A>G>C>U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (?3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.     

ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.

Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.     

AUG codons at the beginning of protein coding sequences are frequent in eukaryotic mRNAs with a suboptimal start codon context

MOTIVATION: The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. However, mRNAs with a suboptimal context of start AUG codon are relatively abundant. It is likely that at least some mRNAs with suboptimal start codon context contain the other signals providing additional information for efficient AUG recognition. RESULTS: Frequency of AUG codons at the beginning of the coding part of eukaryotic mRNAs was analyzed in relation to the context of translation start codon. It was found that the observed downstream AUG content in the mRNAs with optimal start codon context was close to the expected value, whereas it was significantly higher in the mRNAs with a suboptimal context. It is likely that downstream AUG codons can often be utilized as additional start sites to increase translation rate of mRNAs with a suboptimal context of the annotated start codon and many eukaryotic proteins can be characterized by some N-end heterogeneity.