Mechanisms of the initiation of protein synthesis: in reading frame binding of ribosomes to mRNA

The various mechanisms proposed to describe the initiation of protein synthesis are reviewed with a focus on their initiation signals. A characteristic feature of the various mechanisms is that each one of them postulates a distinct initiation signal. The signals of the Shine–Dalgarno (SD), the scanning and the internal ribosome entry site (IRES) mechanisms are all located exclusively in the 5′ leader sequence, whereas, the signal of the cumulative specificity (CS) mechanism includes the entire initiation site (IS). Computer analysis of known E. coli IS sequences showed signal characteristics in the entire model IS consisting of 47 bases, in segments of the 5′ leader and of the protein-coding regions. The proposal that eukaryotic translation actually occurs in two steps is scrutinized. In a first step, initiation factors (eIF4F) interact with the cap of the mRNA, thereby enhancing the accessibility of the IS. In the second step, initiation is by the conserved prokaryotic mechanism in which the ribosomes bind directly to the mRNA without ribosomal scanning. This binding occurs by the proposed process of in reading frame binding of ribosomes to mRNA, which is consistent with the CS mechanism. The basic CS mechanism is able to account for the initiation of translation of leaderless mRNAs, as well as for that of canonical mRNAs. The SD, the scanning and the IRES mechanisms, on the other hand, are inconsistent with the initiation of translation of leaderless mRNAs. Based on these and other observations, it is deemed that the CS mechanism is the universal initiation mechanism.     

A unified view of the initiation of protein synthesis

The mechanism of the initiation of protein synthesis is discussed in terms of two different hypotheses in which each emphasized a different possible element of the process: the Shine-Dalgarno (SD) hypothesis ascribed an essential role to recognition of the SD segment by the ribosomal RNA; it is supported by a variety of experiments but conflicting evidence negates its obligatory nature. In contrast, our hypothesis highlighted the role of the structure of the mRNA and proposes that the initiation codon is selected by virtue of its unique accessibility. The rationale for the importance of accessibility in the selection of the initiation site is discussed. An analysis and a recapitulation of the initiation process and ribosomal specificity are presented. The apparent conflicts with the SD hypothesis are resolved in a unified mechanism where accessibility is the dominant factor.     

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.     

The plant translational apparatus

Protein synthesis in both eukaryotic and prokaryotic cells is a complex process requiring a large number of macromolecules: initiation factors, elongation factors, termination factors, ribosomes, mRNA, amino-acylsynthetases and tRNAs. This review focuses on our current knowledge of protein synthesis in higher plants.Abbreviations eIF eukaryotic initiation factor - eEF eukaryotic elongation factor - EST expressed sequence tag - eRF eukaryotic release factor - GUS -glucoronidase - HCR heme-controlled repressor - PKR double-stranded - RNA activated protein kinase - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Binding of inosine-substituted mRNA to reticulocyte ribosomes and eukaryotic initiation factors 4A and 4B requires ATP

We examined the hypothesis that initiation of eukaryotic protein synthesis involves ATP-dependent melting of 5'-cap-proximal secondary structure in mRNA by eukaryotic initiation factors 4A and 4B. In reticulocyte lysate depleted of ribonucleoside triphosphates by pretreatment with hexokinase/glucose, initiation complex formation by native reovirus mRNA showed a strict requirement for ATP. The corresponding mRNA synthesized with ITP in place of GTP to minimize secondary structure also required ATP for binding to 40 S ribosomal subunits in complexes characteristic of initiation. In a partial reaction without ribosomes, purified eukaryotic initiation factors 4A and 4B bound and cross-linked to the capped 5'-end of oxidized mRNA. This interaction was ATP-dependent with inosine-substituted or bromouridine-containing reovirus RNAs as observed previously with native mRNA. The results indicate that if initiation involves ATP-dependent denaturation of mRNA, the effect must occur after initiation factor-mediated attachment of mRNA to the 40 S ribosomal subunit.     

Ribosomal proteins at the stalk region modulate functional rRNA structures in the GTPase center

Replacement of the L10.L7/L12 protein complex and L11 in Escherichia coli ribosomes with the respective rat counterparts P0.P1/P2 and eukaryotic L12 causes conversion of ribosomal specificity for elongation factors from prokaryotic elongation factor (EF)-Tu/EF-G to eukaryotic EF (eEF)-1alpha/eEF-2. Here we have investigated the effects of protein replacement on the structure and function of two rRNA domains around positions 1070 and 2660 (sarcin/ricin loop) of 23 S rRNA. Protein replacement at the 1070 region in E. coli 50 S subunits was demonstrated by chemical probing analysis. Binding of rat proteins to the 1070 region caused increased accessibility of the 2660 and 1070 regions to ligands for eukaryotic ribosomes: the ribotoxin pepocin for the 2660 region (E. coli numbering), anti-28 S autoantibody for the 1070 region, and eEF-2 for both regions. Moreover, binding of the E. coli L10.L7/L12 complex and L11 to the 1070 region was shown to be responsible for E. coli ribosomal accessibility to another ribotoxin, gypsophilin. Ribosomal proteins at the 1070 region appear to modulate the structures and functions of the 2660 and 1070 RNA regions in slightly different modes in prokaryotes and eukaryotes.     

Dissecting functional similarities of ribosome-associated chaperones from Saccharomyces cerevisiae and Escherichia coli

Ribosome-tethered chaperones that interact with nascent polypeptide chains have been identified in both prokaryotic and eukaryotic systems. However, these ribosome-associated chaperones share no sequence similarity: bacterial trigger factors (TF) form an independent protein family while the yeast machinery is Hsp70-based. The absence of any component of the yeast machinery results in slow growth at low temperatures and sensitivity to aminoglycoside protein synthesis inhibitors. After establishing that yeast ribosomal protein Rpl25 is able to recruit TF to ribosomes when expressed in place of its Escherichia coli homologue L23, the ribosomal TF tether, we tested whether such divergent ribosome-associated chaperones are functionally interchangeable. E. coli TF was expressed in yeast cells that lacked the endogenous ribosome-bound machinery. TF associated with yeast ribosomes, cross-linked to yeast nascent polypeptides and partially complemented the aminoglycoside sensitivity, demonstrating that ribosome-associated chaperones from divergent organisms share common functions, despite their lack of sequence similarity.     

Analysis of ribosome binding sites from the s1 message of reovirus: Initiation at the first and second AUG codons

Two ribosome-protected initiation sites from the s1 message of reovirus have been characterized. Comparison of these sites with the previously determined sequence of s1 mRNA (Li et al., 1980) reveals that wheat germ ribosomes select and protect the first two AUG triplets in that message. This is unusual, since ribosomes initiate at a single site, the 5′-proximal AUG, in almost all other eukaryotic messenger RNAs that have been examined. The first AUG codon in s1 mRNA is preceded by a pyrimidine in position ?3, thus distinguishing it from most other eukaryotic messages, which have a purine (usually A) in that position. The behavior of s1 mRNA is consistent with the hypothesis that flanking nucleotides modulate the efficiency with which the migrating 40 S ribosomal subunit recognizes an AUG codon as a stop signal. If the first AUG triplet is flanked by suboptimal sequences, as in s1 mRNA, some 40 S ribosomes bypass that site and initiate at the next AUG downstream. The second AUG in the s1 message conforms to the consensus sequence (A-N-N-A-U-G-G) for eukaryotic initiation sites.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

The ribosomal stalk is required for ribosome binding,depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α‐sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild‐type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild‐type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.     

The Yeast Omnipotent Suppressor Sup46 Encodes a Ribosomal Protein Which Is a Functional and Structural Homolog of the Escherichia Coli S4 Ram Protein

The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.     

Structural basis for selectivity and toxicity of ribosomal antibiotics

Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents. Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis. Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug. This has important implications for drug specificity and toxicity. Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design.     

Investigation of structure of the ribosomal L12/P stalk

This review contains recent data on the structure of the functionally important ribosomal domain, L12/P stalk, of the large ribosomal subunit. It is the most mobile site of the ribosome; it has been found in ribosomes of all living cells, and it is involved in the interaction between ribosomes and translation factors. The difference between the structures of the ribosomal proteins forming this protuberance (despite their general resemblance) determines the specificity of interaction between eukaryotic and prokaryotic ribosomes and the respective protein factors of translation. In this review, works on the structures of ribosomal proteins forming the L12/P-stalk in bacteria, archaea, and eukaryotes and data on structural aspects of interactions between these proteins and rRNA are described in detail.     

Eukaryotic ribosomal proteins lacking a eubacterial counterpart: important players in ribosomal function

The ribosome is a macromolecular machine responsible for protein synthesis in all organisms. Despite the enormous progress in studies on the structure and function of prokaryotic ribosomes, the respective molecular details of the mechanism by which the eukaryotic ribosome and associated factors construct a polypeptide accurately and rapidly still remain largely unexplored. Eukaryotic ribosomes possess more RNA and a higher number of proteins than eubacterial ribosomes. As the tertiary structure and basic function of the ribosomes are conserved, what is the contribution of these additional elements? Elucidation of the role of these components should provide clues to the mechanisms of translation in eukaryotes and help unravel the molecular mechanisms underlying the differences between eukaryotic and eubacterial ribosomes. This article focuses on a class of eukaryotic ribosomal proteins that do not have a eubacterial homologue. These proteins play substantial roles in ribosomal structure and function, and in mRNA binding and nascent peptide folding. The role of these proteins in human diseases and viral expression, as well as their potential use as targets for antiviral agents is discussed.     

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum phosphofructokinase

The nucleotide sequence of a full-length cDNA encoding phosphofructokinase (PFK) enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2,653 bases comprises a single open reading frame of 2,452 bases and a noncoding region of 201 bases after the stop codon. The mature protein contains 812 amino acids and has a molecular mass of 90,900 Da. The amino acid sequences of several peptides derived from the purified protein show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the protein with those of 3 other worms as well as those of human, rabbit, and bacterial enzymes reveals highly conserved regions interrupted with stretches of lesser sequence similarity. Analyses of the subunit primary structure reveal, as in other eukaryotic PFKs, that the amino-terminal half is homologous to the carboxy-terminal half, supporting the hypothesis that the PFK gene evolved by duplication of the prokaryotic gene and that the allosteric sites arose by mutations at the catalytic site. The location of the phosphorylation site is unique and different compared with other PFKs and plays a key role in regulation of the enzyme activity. Structural motifs such as the putative substrate and effector binding domains and also the key amino acids involved therein are clearly identified by alignment of all the PFK protein sequences.     

Stage-specific initiation factors for protein synthesis during insect development

In insects, as in bacteria, the smaller (40 S) ribosomal subunit binds messenger RNA during initiation of protein synthesis. An 80 S ribosomal unit is formed by association of free 40 S and 60 S subunits. Formation of the complete initiation complex requires GTP, aminoacyl-tRNA, protein initiation factors and messenger RNA. The complex sediments as an 80 S band on sucrose gradient. Protein initiation factors are extracted from unwashed ribosomes and appear to be able to discriminate between messenger RNAs obtained from different stages of development. They promote formation of the 80 S complex only when messenger RNA is extracted from the same stage of development, providing a mechanism for control of protein synthesis by which ribosomes can select the messenger RNA to be translated. Two possibilities have been proposed to explain this phenomenon: (1) that a group of messenger RNAs from a given stage of development may have a specific sequence of nucleotides preceding the AUG codon. This sequence is recognized by a stage-specific element of the initiation machinery; (2) and or, the secondary structure of messenger RNA from a given stage of development may be specific and therefore recognized by a unique initiation factor.