Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs.

The nucleotide sequence of the translation initiation regions of 96 Saccharomyces cerevisiae mRNAs was compiled and compared. The entire 5' untranslated sequence of most mRNAs is very rich in A-residues. G-residues are underrepresented in the untranslated region. The AUG startcodon context appeared to be distinctly different from that of animal mRNAs, although an A-residue at -3 also occurs very frequently (81 percent) in yeast mRNAs. The prevailing codon 3' adjacent to the AUG is the UCU serine codon. All these features are more extreme in the highly expressed genes. Fifty percent of all highly expressed genes use the UCU serine codon as second triplet. In this group G-residues are completely absent in the 7 bases preceding the startcodon and an A-residue occurs at position -1 and -3 at a frequency of 89 percent and 100 percent, respectively. The abundance of A-residues throughout the leader suggests that unstructured mRNA is required for efficient translation initiation in yeast. The consensus sequence for the AUG context in highly expressed genes can be summarized as follows: (Sequence: see text).     

Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates.

The previously presented consensus sequence for eukaryotic translation initiation sites by Kozak was derived substantially from vertebrate mRNA sequences. Drosophila nuclear genes exhibit a significantly different translation start consensus sequence. These differences probably do not represent mechanistic differences in translation initiation inasmuch as both taxa exhibit identical preferences and restrictions at the crucial -3 position. Using more conservative criteria for the assignment of consensus the following consensus sequences were derived: vertebrate--CANCAUG and Drosophila--CAAAACAUG.     

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.

5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.     

At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells

Sequences flanking the AUG initiator codon influence its recognition by eukaryotic ribosomes. From a comparison of several hundred mRNA sequences, CCA/GCCAUGG emerged as the consensus sequence for initiation in higher eukaryotes. Systematic mutagenesis of a cloned preproinsulin gene confirmed the facilitating effect of A or G in position -3 (i.e. 3 nucleotides upstream from the AUG codon), C in positions -1 and -2, and G immediately following the AUG codon. The analysis of a new set of mutants now reveals that sequences slightly farther upstream are also influential, the optimal context for initiation being (GCC)GCCA/GCCAUGG. Possible mechanistic implications of the repeating GCC motif are discussed.     

Specific interactions of histone H1 and a 45 kilodalton nuclear protein with a putative matrix attachment site in the distal promoter region of a cell cycle-regulated human histone gene

Protein-DNA interactions within the promoter of a cell cycle-regulated human H4 histone gene were examined by binding of 5'-end-labeled DNA segments to Western blots of nuclear protein fractions. Specific protein interactions were observed with DNA segments located between -500 bp and -1,070 bp upstream of the ATG initiation codon and included a histone H1 binding segment flanked on both sides by binding sites for a 45 kD nuclear protein. This region of the gene contains a DNase I-sensitive site in the center (-720 to -820 bp), and sequence analysis revealed the presence of scaffold attachment sequences in the two flanking segments. Topoisomerase II consensus sequences and in vitro topoisomerase II cleavage sites were also detected in the two flanking segments. Our results suggest that the 45 kd nuclear protein may preferentially interact with these two segments of the H4 histone gene to mediate association with the nuclear matrix. The presence of negative regulatory elements in this putative matrix attachment region provides a basis for the speculation that such nuclear proteins are associated with alterations in gene-matrix interaction that are functionally related to gene expression.     

Correlation between sequence conservation of the 5' untranslated region and codon usage bias in Mus musculus genes

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.     

Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes

By analyzing the effects of single base substitutions around the ATG initiator codon in a cloned preproinsulin gene, I have identified ACCATGG as the optimal sequence for initiation by eukaryotic ribosomes. Mutations within that sequence modulate the yield of proinsulin over a 20-fold range. A purine in position -3 (i.e., 3 nucleotides upstream from the ATG codon) has a dominant effect; when a pyrimidine replaces the purine in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Single base substitutions around an upstream, out-of-frame ATG codon affect the efficiency with which it acts as a barrier to initiating at the downstream start site for preproinsulin. The optimal sequence for initiation defined by mutagenesis is identical to the consensus sequence that emerged previously from surveys of translational start sites in eukaryotic mRNAs. The mechanism by which nucleotides flanking the ATG codon might exert their effect is discussed.     

Vertebrate histone genes: nucleotide sequence of a chicken H2A gene and regulatory flanking sequences.

The DNA sequence of a chicken genomal fragment containing a histone H2A gene has been determined. It contains extensive 5' and 3' flanking regions and encodes a protein identical in sequence to the histone H2A protein isolated from chicken erythrocytes. In the 5' flanking region, a possible "TATA box" and three possible "cap sites" can be recognised upstream from the initiation codon. To the 5' side of the "TATA box" is found an unusual sequence of 21 A's interrupted by a central G residue. It occupies the same relative position as the P. miliaris H2A gene-specific 5' dyad symmetry sequence and the "CCAAT box" seen in other eukaryotic polymerase II genes but is clearly different from both. A significant feature of the 3' non-coding region is the presence of a 23 base-pair sequence that is nearly identical to a conserved region found in sea urchin histone genes. The coding region is extremely GC rich, with strong selection for these bases in the third position of codons. Not a single coding triplet ends in U. No intervening sequences were found in this gene.