Evidence for a Competitive-Displacement Model for the initiation of protein synthesis involving the intermolecular hybridization of 5 S rRNA, 18 S rRNA and mRNA.

We have previously shown that a 5'-terminal region of mouse 5 S rRNA can base-pair in vitro with two distinct regions of 18 S rRNA. Further analysis reveals that these 5 S rRNA-complementary sequences in 18 S rRNA also exhibit complementarity to the Kozak consensus sequence surrounding the mRNA translational start site. To test the possibility that these 2 regions in 18 S rRNA may be involved in mRNA binding and translational initiation, we have tested, using an in vitro translation system, the effects of DNA oligonucleotides complementary to these 18 S rRNA sequences on protein synthesis. Results show that an oligonucleotide complementary to one 18 S rRNA region does inhibit translation at the step of initiation. We propose a Competitive-Displacement Model for the initiation of translation involving the intermolecular base-pairing of 5 S rRNA, 18 S rRNA and mRNA.     

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.     

Selection of AUG initiation codons differs in plants and animals.

The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.     

Conservation of translation initiation sites based on dinucleotide frequency and codon usage in Escherichia coli K-12 (W3110): non-random distribution of A/T-rich sequences immediately upstream of the translation initiation codon.

Dinucleotide frequencies are useful for characterizing consensus elements as a minimum unit of nucleotide sequence because the neighborhood relations of nucleotide sequences are reflected in dinucleotides. Using a consensus score based on dinucleotide frequencies and intra-species codon usage heterogeneity, denoted by the Z1 parameter, we report the relationship between nucleotide conservation at the translation initiation sites of genes in the Escherichia coli K-12 genome (W3110) and codon usage in its downstream genes. Significant positive correlations were obtained in three regions centered at -13, -4, and +7, which correspond to the Shine-Dalgarno element, the A + T element immediately upstream of the translation initiation site, and the downstream box, respectively.     

Translation of chloroplast-encoded mRNA: potential initiation and termination signals.

A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively. As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22. A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes. In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation. A SD sequence could not be located in 6% of the chloroplast sequences. We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation. We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E. coli.     

Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes

Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.     

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

We determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine–Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5'-AGGA-3' to 5'-UUUU-3' results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon–anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon–anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.     

Structural model for the selenocysteine-specific elongation factor SelB

A structural model was established for the N-terminal part of translation factor SelB which shares sequence similarity with EF-Tu, taking into account the coordinates of the EF-Tu 3D structure and the consensus of SelB sequences from four bacteria. The model showed that SelB is homologous in its N-terminal domains over all three domains of EF-Tu. The guanine nucleotide binding site and the residues involved in GTP hydrolysis are similar to those of EF-Tu, but with some subtle differences possibly responsible for the higher affinity of SelB for GTP compared to GDP. In accordance, the EF-Tu epitopes interacting with EF-Ts are lacking in SelB. Information on the formation of the selenocysteyl-binding pocket is presented. A phylogenetic comparison of the SelB domains homologous to EF-Tu with those from EF-Tu and initiation factor 2 indicated that SelB forms a separate class of translation factors.     

Normal variants of human mitochondrial DNA and translation products: the building of a reference data base

Summary A good standard reference for the highly polymorphic human mitochondrial DNA (mtDNA) sequence is essential for studies of normal and disease-related nucleotide variants in the mitochondrial genome. A consensus sequence for the human mitochondrial genome has been derived from thirteen unrelated mtDNA sequences. We report 128 nucleotide variants of the human mtDNA sequence, and 62 amino acid variants of the human mitochondrial translation products, observed in the coding region of these mtDNA sequences.     

Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli

The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.     

Escherichia coli mRNAs with strong Shine/Dalgarno sequences also contain 5' end sequences complementary to domain # 17 on the 16S ribosomal RNA

A well-established feature of the translation initiation region, which attracts the ribosomes to the prokaryotic mRNAs, is a purine rich area called Shine/Dalgarno sequence (SD). There are examples of various other sequences, which despite having no similarity to an SD sequence are capable of enhancing and/or initiating translation. The mechanisms by which these sequences affect translation remain unclear, but a base pairing between mRNA and 16S ribosomal RNA (rRNA) is proposed to be the likely mechanism. In this study, using a computational approach, we identified a non-SD signal found specifically in the translation initiation regions of Escherichia coli mRNAs, which contain super strong SD sequences. Nine of the 11 E. coli translation initiation regions, which were previously identified for having super strong SD sequences, also contained six or more nucleotides complementary to box-17 on the 16S rRNA (nucleotides 418-554). Mutational analyses of those initiation sequences indicated that when complementarity to box-17 was eliminated, the efficiency of the examined sequences to mediate the translation of chloramphenicol acetyltransferase (CAT) mRNA was reduced. The results suggest that mRNA sequences with complementarity to box-17 of 16S rRNA may function as enhancers for translation in E. coli.     

Inefficient translation of T7 late mRNA by Bacillus subtilis ribosomes. Implications for species-specific translation

Bacillus subtilis 30 S subunits inefficiently recognize initiation sites in mRNAs from Gram-negative bacteria, but they are able to efficiently recognize initiation sites in mRNA derived from Gram-positive bacteria. McLaughlin et al. (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291) have suggested that B. subtilis ribosomes require a strong Shine-Dalgarno sequence for translation initiation. To test whether this criterion is sufficient to explain the translational specificity of B. subtilis ribosomes, T7 late mRNA, which contains strong Shine-Dalgarno sequences before many of the late genes (Dunn, J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535), was translated in vitro with both Escherichia coli and B. subtilis ribosomes. The identification of several of the in vitro products upon gel electrophoresis indicated that B. subtilis ribosomes recognize correct translation initiation sites in late T7 mRNA, but they do not translate these products efficiently. Competition experiments demonstrated that late T7 mRNA does not inhibit B. subtilis ribosomal translation of B. subtilis derived mRNA (from the bacteriophage phi 29). It is concluded that strong Shine-Dalgarno sequences may be necessary in B. subtilis translation initiation sites; however, additional determinants of initiation which differ from those found in the translation initiation sites of E. coli mRNAs must exist.     

Sequence analysis of rice rubi3 promoter gene expression cassettes for improved transgene expression

In a construct containing a GUS reporter gene driven by the 5′ regulatory elements from rubi3, expression was enhanced 4-fold when a 20-nucleotide (nt) GUS 5′ untranslated sequence was replaced with 9 nt sequences derived from rubi3′s second exon. The roles of the sequences immediately upstream from the GUS translation initiation codon, and their significance in gene expression, were investigated. Sequence analysis suggests that complementarity between sequences immediately 5′ of a translation initiation codon and the rice 17S rRNA may be responsible for the reduction in protein levels from constructs containing the GUS leader sequence. The results demonstrate an affect sequences immediately upstream from transgenic coding sequences have on expression, and when using the rubi3 5′ regulatory sequence in particular.