Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation.

The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII gene is located in the leftward operon, which is transcribed from the pL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression.     

RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5′-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5′-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine–Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.     

Translational repression of the Escherichia coli alpha operon mRNA: importance of an mRNA conformational switch and a ternary entrapment complex

Ribosomal protein S4 represses synthesis of the four ribosomal proteins (including itself) in the Escherichia coli alpha operon by binding to a nested pseudoknot structure that spans the ribosome binding site. A model for the repression mechanism previously proposed two unusual features: (i) the mRNA switches between conformations that are "active" or "inactive" in translation, with S4 as an allosteric effector of the inactive form, and (ii) S4 holds the 30 S subunit in an unproductive complex on the mRNA ("entrapment"), in contrast to direct competition between repressor and ribosome binding ("displacement"). These two key points have been experimentally tested. First, it is found that the mRNA pseudoknot exists in an equilibrium between two conformers with different electrophoretic mobilities. S4 selectively binds to one form of the RNA, as predicted for an allosteric effector; binding of ribosomal 30 S subunits is nearly equal in the two forms. Second, we have used S4 labeled at a unique cysteine with either of two fluorophores to characterize its interactions with mRNA and 30 S subunits. Equilibrium experiments detect the formation of a specific ternary complex of S4, mRNA pseudoknot, and 30 S subunits. The existence of this ternary complex is unambiguous evidence for translational repression of the alpha operon by an entrapment mechanism.     

Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA

We have previously shown that stable base-pairing at a translational initiation site in Escherichia coli can inhibit translation by competing with the binding of ribosomes. When the base-pairing is not too strong, this competition is won by the ribosomes, resulting in efficient translation from a structured ribosome binding site (RBS). We now re-examine these results in the light of RNA folding kinetics and find that the window during which a folded RBS is open is generally much too short to recruit a 30S ribosomal subunit from the cytoplasm. We argue that to achieve efficient expression, a 30S subunit must already be in contact with the mRNA while this is still folded, to shift into place as soon as the structure opens. Single-stranded regions flanking the structure may constitute a standby site, to which the 30S subunit can attach non-specifically. We propose a steady-state kinetic model for the early steps of translational initiation and use this to examine various quantitative aspects of standby binding. The kinetic model provides an explanation of why the earlier equilibrium competition model predicted implausibly high 30S-mRNA affinities. Because all RNA is structured to some degree, standby binding is probably a general feature of translational initiation.     

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.     

Direct ribosomal binding by a cellular inhibitor of translation

During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation while allowing continued translation of specific messenger RNAs encoding regulatory and stress-response proteins. We report here that the apoptotic regulator Reaper inhibits protein synthesis by binding directly to the 40S ribosomal subunit. This interaction does not affect either ribosomal association of initiation factors or formation of 43S or 48S complexes. Rather, it interferes with late initiation events upstream of 60S subunit joining, apparently modulating start-codon recognition during scanning. CrPV IRES-driven translation, involving direct ribosomal recruitment to the start site, is relatively insensitive to Reaper. Thus, Reaper is the first known cellular ribosomal binding factor with the potential to allow selective translation of mRNAs initiating at alternative start codons or from certain IRES elements. This function of Reaper may modulate gene expression programs to affect cell fate.     

Meanderings of the mRNA through the ribosome

A map of how mRNA travels through the ribosome is critical for any detailed understanding of the process of translation. This feat has recently been achieved using X-ray crystallography. The structure reveals, for the first time, details of the interactions between the mRNA and the 30S subunit beyond those at the tRNA binding sites. Elements of both 16S rRNA and ribosomal proteins contribute to mRNA binding. This work also identifies two tunnels that the mRNA passes through as it wraps around the 30S subunit. The mechanisms and mechanics of reading frame selection, translational fidelity, and translocation can now be informed by the structure.     

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a ΔS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA^fMet, and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.     

Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless cI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2. Here, we have studied this phenomenon at the molecular level by making use of an E. coli rpsB(ts) mutant. The analysis of the ribosomes isolated under the non-permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit. In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non-permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA gene, encoding the "DeaD/CsdA" DEAD-box helicase, has been isolated as a multicopy suppressor of rpsB(ts) mutations. Here, we show that expression of a plasmid-borne DeaD/CsdA gene restores both S1 and S2 on the ribosome at the non-permissive temperature in the rpsB(ts) strain, which in turn leads to suppression of the translational defect affecting canonical mRNSa. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.     

Interactions of the non-coding RNA DsrA and RpoS mRNA with the 30 S ribosomal subunit

Expression of sigma(s), the gene product of rpoS, is controlled translationally in response to many environmental stresses. DsrA, a small 87-nucleotide non-coding RNA molecule, acts to increase translational efficiency of RpoS mRNA under some growth conditions. In this work, we demonstrate that DsrA binds directly to the 30 S ribosomal subunit with an observed equilibrium affinity of 2.8 x 10(7) m(-1). DsrA does not compete with RpoS mRNA or tRNA(f)(Met) for binding to the 30 S subunit. The 5' end of DsrA binds to 30 S subunits with an observed equilibrium association constant of 2.0 x 10(6) m(-1), indicating that the full affinity of the interaction requires the entire DsrA sequence. In order to investigate translational efficiency of RpoS mRNA, we examined both ribosome-binding site accessibility and the binding of RpoS mRNA to 30 S ribosomal subunits. We find that that ribosome-binding site accessibility is modulated as a function of divalent cation concentration during mRNA renaturation and by the presence of an antisense sequence that binds to nucleotides 1-16 of the RpoS mRNA fragment. The ribosome-binding site accessibility correlates with the amount of RpoS mRNA participating in 30 S-mRNA "pre-initiation" translational complex formation and provides evidence that regulation follows a competitive model of regulation.     

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acid involved in its ribosomal binding and recycling

Starting from a synthetic modular gene (infA) encoding Escherichia coli translation initiation factor IF1, we have constructed mutants in which amino acids are deleted from the carboxyl terminus or in which His29 or His34 are replaced by Tyr or Asp residues. The mutant proteins were overproduced, purified and tested in vitro for their properties in several partial reactions of the translation initiation pathway and for their capacity to stimulate MS2 RNA-dependent protein synthesis. The results allow for the conclusion that: (i) Arg69 is part of the 30S ribosomal subunit binding site of IF1 and its deletion results in the substantial loss of all IF1 function; (ii) neither one of its two histidines is essential for the binding of IF1 to the 30S ribosomal subunit, for the stimulation of fMet-tRNA binding to 30S or 70S ribosomal particles or for MS2 RNA-dependent protein synthesis; but (iii) His29 is involved in the 50S subunit-induced ejection of IF1 from the 30S ribosomal subunit.     

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.     

Participation of initiation factor IF-3 in the binding of AcPhe-tRNA to the 30S ribosomal subunit

Initiation factor IF-3 is required in addition to IF-1 and IF-2 for maximal initial rate of poly(U)-directed binding of AcPhe-tRNA to 30S ribosomal subunits of $\text{E. coli}$. Incubation periods longer than 10 sec, by which time the reaction is virtually over, progressively obscure the requirement for IF-3 in AcPhe-tRNA binding. IF-3 also stimulates the poly(A, G, U)-directed binding of fMet-tRNA to the 30S ribosomal subunit, but in this case, significant stimulation can still be observed even with extended incubation. These results indicate that IF-3 functions similarly in the translation of synthetic mRNA, as it does with natural mRNA, participating in ribosome dissociation and in the formation of the initiation complex from the 30S ribosomal subunit.     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.