Constraints on codon context in Escherichia coli genes. Their possible role in modulating the efficiency of translation

The constraints on nucleotide sequences of highly and weakly expressed genes from Escherichia coli have been analysed and compared. Differences in synonymous codon spectra in highly and weakly expressed genes lead to different frequencies of nucleotides (in the first and third codon positions) and dinucleotides in the two groups of genes. It has been found that the choice of synonymous codons in highly expressed genes depends on the nucleotides adjacent to the codon. For example, lysine is preferably encoded by the AAA codon if guanosine is 3' to the lysine codon (AAA-G, P less than 10(-9)). And, on the contrary, AAG is used more often than AAA (P less than 0.001) if cytidine is 3' adjacent to lysine. Guanosine occurs more frequently than adenosine 5' to all the lysine codons (AAR, P less than 10(-5), i.e. NNG codons are preferred over the synonymous NNA codons 5' to the positions of lysine in the genes. The context effect was observed in nonsense and missense suppression experiments. Therefore, a hypothesis has been suggested that the efficiency of translation of some codons (for which the constraints on the adjacent nucleotides were found) can be modulated by the codon context. The rules for preferable synonymous codon choice in highly expressed genes depending on the nucleotides surrounding the codon are presented. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

Partial characterization of bovine elastin gene; comparison with the gene for human elastin

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5' direction from the initiation codon and 978 nucleotides in the 3' direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5' to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.     

Pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2.

Retroviruses whose gag and pol genes are in the same reading frame depend upon approximately 5% read-through of the gag UAG termination codon to make the gag-pol polyprotein. For murine leukemia virus, this read-through is dependent on a pseudoknot located eight nucleotides 3' of the UAG. Other retroviruses whose gag and pol genes are in the same frame can potentially form similar pseudoknots 3' of their UAG codons. Beyond the similar secondary structures, there is strong sequence conservation in the spacer region and in loop 2 of the pseudoknots. The detrimental effects of substitutions of several of these conserved spacer and loop 2 nucleotides in the murine leukemia virus sequence show their importance for the read-through process. The importance of specific nucleotides in loop 2 of the pseudoknot contrasts with the flexibility of sequence in loop 2 of the most intensively studied frameshift-promoting pseudoknot which occurs in infectious bronchitis virus. Two nucleotides in loop 2 of the murine leukemia virus pseudoknot, which were shown to be important by mutagenic analysis, display hypersensitivity to the single-strand specific nuclease, S1. They are likely to be particularly accessible or are in an unusually reactive conformation.     

Quantitative parameters of the 3'-5'-exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 and DNA with single-strand breaks containing dYMP or their modified analogues

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, the cleavage of DNA 5' to the AP site, it displays other weak enzymatic activities. One of them is 3'-5' exonuclease activity, which is most effectively pronounced for DNA duplexes containing modified or mismatched nucleotides at the 3' end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase beta during the base excision repair process. We determined the quantitative parameters of the 3'-5' exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3' terminus of single-strand DNA and from photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control.

Translational control of the GCN4 gene involves two short open reading frames in the mRNA leader (uORF1 and uORF4) that differ greatly in the ability to allow reinitiation at GCN4 following their own translation. The low efficiency of reinitiation characteristic of uORF4 can be reconstituted in a hybrid element in which the last codon of uORF1 and 10 nucleotides 3' to its stop codon (the termination region) are substituted with the corresponding nucleotides from uORF4. To define the features of these 13 nucleotides that determine their effects on reinitiation, we separately randomized the sequence of the third codon and termination region of the uORF1-uORF4 hybrid and selected mutant alleles with the high-level reinitiation that is characteristic of uORF1. The results indicate that many different A+U-rich triplets present at the third codon of uORF1 can overcome the inhibitory effect of the termination region derived from uORF4 on the efficiency of reinitiation at GCN4. Efficient reinitiation is not associated with codons specifying a particular amino acid or isoacceptor tRNA. Similarly, we found that a diverse collection of A+U-rich sequences present in the termination region of uORF1 could restore efficient reinitiation at GCN4 in the presence of the third codon derived from uORF4. To explain these results, we propose that reinitiation can be impaired by stable base pairing between nucleotides flanking the uORF1 stop codon and either the tRNA which pairs with the third codon, the rRNA, or sequences located elsewhere in GCN4 mRNA. We suggest that these interactions delay the resumption of scanning following peptide chain termination at the uORF and thereby lead to ribosome dissociation from the mRNA.     

Development of a somatic mutation screening system using Hb mutants. IV. Successful detection of red cells containing the human frameshift mutants Hb Wayne and Hb Cranston using monospecific fluorescent antibodies.

The production and purification of antibodies detecting Hb Wayne, an alpha-globin frameshift mutant, and Hb Cranston, a beta-globin frameshift mutant, are described. The antibodies are of a nonprecipitating nature, and they permit strong fluorescent labeling of erythrocytes containing Hb Wayne or Hb Cranston. Studies using artificial mixtures containing cells with either of the two mutants in frequencies ranging from 1 in 10(2) to 1 in 10(5) showed that fluorescent antibodies can detect rare mutant red cells in the presence of vast excesses of normal erythrocytes. On the basis of the structures and the molecular lesions underlying production of the two abnormal hemoglobins, we predict that the anti-Hb Wayne antibody will detect several frameshift mutants resulting from deletion of 3n + 1 nucleotides or insertion of 3n + 2 nucleotides at the 5' side of the codon normally specifying residue 139 of the alpha chain. The anti-Hb Cranston antibody should be capable of detecting beta chains, the corresponding genes of which have sustained insertions of 3n + 2 nucleotides or deletions of 3n + 1 nucleotides on the 5' side of the codon normally specifying residue 144. The two antibodies may, therefore, prove to be valuable in the development of a system aimed at detecting rare erythrocytes that express mutations which arise in the hemopoietic stem cells of normal individuals and subjects exposed to mutagens.     

A single nucleotide difference at the 3'' end of an intron causes differential splicing of two histocompatibility genes.

The murine histocompatibility class I genes, H-2 Kb and Kk, display considerable homology at their 3' ends. In fact, from exon 5 to the termination codon, only two nucleotides differ between the two genes, one at the 5' end and the other at the 3' end of intron 7. Despite this similarity, the gene products have distinctly different mol. wts as determined by SDS-PAGE. By constructing two hybrid genes, pC2 and pC4, we demonstrated that it is the cytoplasmic parts of the antigens (encoded by exons 6-8) which are responsible for the major difference in mol. wt. We have used site-directed mutagenesis to change the two nucleotides in intron 7 of the H-2 Kk gene to those present in the H-2 Kb gene. S1 nuclease mapping has been used to identify the actual splice site of the authentic Kb and Kk genes, the hybrid genes and the mutagenized genes. We have shown that it is the 3' nucleotide difference, nine nucleotides upstream of the 3' splice site, which causes the different excision of intron 7 of the Kb gene. The 5' nucleotide difference does not alter the splicing. The choice of branch points and 3' splice signals for intron 7 of five H-2 class I genes, is discussed.     

Analysis of the primary structure of mRNA from Escherichia coli: occurrence of nucleotides on the 3'-side of the codon

The occurrence of nucleotides of the 3' side of codons has been determined in highly and weakly expressed genes from Escherichia coli. It was found that the usage of some amino acid codons in highly expressed genes was site specific, depending on the base 3' to the codon. The role of the 3' nucleotide as a modulator of codon translation effectiveness is discussed. The rules of synonymous codon usage in relation to the 3' flanking nucleotide have been established for highly expressed genes. For example, if a triplet next to the lysine codon starts with guanosine, lysine is preferably encoded by AAA and not by AAG (P less than 10(-8), while of cytidine is 3' to the lysine codon, AAG is preferred over AAA (P less than 0.001). These rules are observed in highly and absent in weakly expressed mRNAs and can be used in the chemical synthesis of genes designed for expression in E. coli.     

Sense codon in Escherichia coli are translated in context

The nucleotide frequencies 5' and 3' to the sense codons in highly and weakly expressed genes have been investigated by the chi-squares method. A comparison between the experimental and computer-generated random nucleotide sequences (in which each codon is substituted by a random synonymous one) was made. It was shown that the choice of a particular codon among the synonymous ones in a given position of the gene depends on the three nucleotides 3' and 5' adjacent to the codon in highly expressed genes (the triplet 3' and a single nucleotide 5' to the codons in weakly expressed genes). Concrete patterns for the preferable choice of synonymous codons depending on their contexts are presented. It is suggested that these constraints are related to the efficiency of messenger translation. The constraints on the amino acid sequences of encoded proteins also lead to statistically significant bases in nucleotide frequencies around the sense codons. The biological role of these constraints is discussed.     

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.     

Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus.

Retroviruses, such as murine leukemia virus (MuLV), whose gag and pol genes are in the same reading frame but separated by a UAG stop codon, require that 5-10 % of ribosomes decode the UAG as an amino acid and continue translation to synthesize the Gag-Pol fusion polyprotein. A specific pseudoknot located eight nucleotides 3' of the UAG is required for this redefinition of the UAG stop codon. The structural probing and mutagenic analyses presented here provide evidence that loop I of the pseudoknot is one nucleotide, stem II has seven base-pairs, and the nucleotides 3' of stem II are important for function. Stem II is more resistant to single-strand-specific probes than stem I. Sequences upstream of the UAG codon allow formation of two competing structures, a stem-loop and the pseudoknot.     

Consecutive GA pairs stabilize medium-size RNA internal loops

Internal loops in RNA are important for folding and function. Consecutive noncanonical pairs can form in internal loops having at least two nucleotides on each side. Thermodynamic and structural insights into such internal loops should improve approximations for their stabilities and predictions of secondary and three-dimensional structures. Most natural internal loops are purine rich. A series of oligoribonucleotides that form purine-rich internal loops of 5-10 nucleotides, including kink-turn loops, were studied by UV melting, exchangeable proton and phosphorus NMR. Three consecutive GA pairs with the motif 5' Y GGA/3' R AAG or GGA R 3'/AAG Y 5' (i.e., 5' GGA 3'/3' AAG 5' closed on at least one side with a CG, UA, or UG pair with Y representing C or U and R representing A or G) stabilize internal loops having 6-10 nucleotides. Certain motifs with two consecutive GA pairs are also stabilizing. In internal loops with three or more nucleotides on each side, the motif 5' U G/3' G A has stability similar to 5' C G/3' G A. A revised model for predicting stabilities of internal loops with 6-10 nucleotides is derived by multiple linear regression. Loops with 2 x 3 nucleotides are predicted well by a previous thermodynamic model.     

The nucleotide sequence of tobacco rattle virus RNA-2 (CAM strain).

The nucleotide sequence of the smaller genomic strand (RNA-2) of the bipartite tobacco rattle virus (CAM strain) has been determined. RNA-2 is capped at the 5' terminus and contains 1799 nucleotide residues. There is a single 223 codon long open reading frame extending from nucleotide 574 to 1242 which designates a protein of Mr 23,654. The derived amino acid composition, in percent, matches that previously determined for the virus capsid protein. The long open reading frame is flanked by 5' and 3' untranslated regions of 573 and 554 nucleotides, respectively. The 5' leader sequence contains two different sets of direct repeats, one of 119 nucleotides and the other of 76. It also contains 13 apparently unused AUG codons, four of which lie in the same frame as the capsid protein cistron. The 3' terminal sequence of RNA-2 is identical to that of the larger genomic strand (RNA-1) for 459 nucleotides.     

A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3

We describe a new computer program, SnpEff, for rapidly categorizing the effects of variants in genome sequences. Once a genome is sequenced, SnpEff annotates variants based on their genomic locations and predicts coding effects. Annotated genomic locations include intronic, untranslated region, upstream, downstream, splice site, or intergenic regions. Coding effects such as synonymous or non-synonymous amino acid replacement, start codon gains or losses, stop codon gains or losses, or frame shifts can be predicted. Here the use of SnpEff is illustrated by annotating ~356,660 candidate SNPs in ~117 Mb unique sequences, representing a substitution rate of ~1/305 nucleotides, between the Drosophila melanogaster w(1118); iso-2; iso-3 strain and the reference y(1); cn(1) bw(1) sp(1) strain. We show that ~15,842 SNPs are synonymous and ~4,467 SNPs are non-synonymous (N/S ~0.28). The remaining SNPs are in other categories, such as stop codon gains (38 SNPs), stop codon losses (8 SNPs), and start codon gains (297 SNPs) in the 5'UTR. We found, as expected, that the SNP frequency is proportional to the recombination frequency (i.e., highest in the middle of chromosome arms). We also found that start-gain or stop-lost SNPs in Drosophila melanogaster often result in additions of N-terminal or C-terminal amino acids that are conserved in other Drosophila species. It appears that the 5' and 3' UTRs are reservoirs for genetic variations that changes the termini of proteins during evolution of the Drosophila genus. As genome sequencing is becoming inexpensive and routine, SnpEff enables rapid analyses of whole-genome sequencing data to be performed by an individual laboratory.     

NMR solution structures of bistranded abasic site lesions in DNA

Ionizing radiation produces clustered lesions in DNA. Since the orientation of bistranded lesions affects their recognition by DNA repair enzymes, clustered damages are more difficult to process and thus more toxic than single oxidative lesions. In order to understand the structural determinants that lead to differential recognition, we used NMR spectroscopy and restrained molecular dynamics to solve the structure of two DNA duplexes, each containing two stable abasic site analogues positioned on opposite strands of the duplex and staggered in the 3' (-1 duplex, (AP) 2-1 duplex) or 5' (+1 duplex, (AP) 2+1 duplex) direction. Cross-peak connectivities observed in the nonexchangeable NOESY spectra indicate compression of the helix at the lesion site of the duplexes, resulting in the formation of two abasic bulges. The exchangeable proton spectra show the AP site partner nucleotides forming interstrand hydrogen bonds that are characteristic of a Watson-Crick G.C base pairs, confirming the extra helical nature of the AP residues. Restrained molecular dynamics simulations generate a set of converging structures in full agreement with the spectroscopic data. In the (AP) 2-1 duplex, the extra helical abasic site residues reside in the minor groove of the helix, while they appear in the major groove in the (AP) 2+1 duplex. These structural differences are consistent with the differential recognition of bistranded abasic site lesions by human AP endonuclease.     

Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

Starting with the lambda pRE-strain lambda ctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, lambda dya2 ctr1 cy3008 and lambda dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations in cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'. --The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII-mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A----G mutation four bases before the initial AUG codon, and cII3059, a GUU----GAU (Val2----Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regularities of the nucleotide sequence at the 5'-end of the codon in Escherichia coli genes

The frequencies of occurrence of nucleotides at the 5' side of codons have been determined in highly and weakly expressed genes from E. coli. Significant constraints on the nucleotide 5' to some codons were found in highly expressed genes. Certain rules of synonymous codon usage depending on the amino acid 3' of the codon were established. E. g., codon possessing quanosine in the third position (NNG) are preferred over NNA if the next amino acid is lysine (P less than 10(-5)). On the other hand, rules of synonymous codon usage in relation to 5' flanking nucleotide were found. For example, when coding for aspartic acid, GAC codon is preferred over GAU (P less than 0.001) if uridine is 5' to codon and on the contrary GAU is favoured (P less than 0.0001) if quanosine is at the 5' side of aspartic acid codon. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.     

The actin gene in yeast Saccharomyces cerevisiae: 5'' and 3'' end mapping, flanking and putative regulatory sequences

The 5' and 3' flanking regions of the yeast actin gene have been sequenced and the ends of the actin mRNA were determined by the single-strand nuclease mapping procedure. The mRNA starts with a pyrimidine residue 141 (or 140) nucleotides upstream from the initiation codon. The actin gene lacks a typical "TATA" box 30 base pairs upstream from the mRNA start site but it contains a region homologous to the canonical sequence 5'-GGCTCAATCT-3' which is found in several eukaryotic genes 70 to 80 bp upstream from the mRNA cap site. Judging from the S1 nuclease mapping, there are two populations of actin mRNA terminating 98 and 107 nucleotides downstream from the stop codon. The 3' termini are preceded by three AATAAA sequences found in most eukaryotic polyadenylated mRNAs.