Efficient expression of gene variants that harbour AGA codons next to the initiation codon

Abstract

In an effort to improve the knowledge about the rules which direct the effect of the early ORF sequences on translation efficiency, we have analyzed the effect of pairs of the six arginine codons at the second and third positions on the expression of lacZ variants. Whereas the pairs of identical AGA or AGG codons were favorable for the gene expression, identical pairs of each of the four CGN codons were very inefficient. This result was unexpected because tandems of AGA or AGG codons located in more internal gene positions provoke deficient expression whilst internally located CGU and CGC are the most abundant and efficiently translated arginine codons. The mixed combinations of AGA and each of the CGN codons usually resulted in efficient rates of lacZ expression independently of the peptidyl-tRNA propensity to dissociate from the ribosome. Thus, the variant harboring the pair of AGA codons was expressed as efficiently as the variant carrying a pair of AAA codons in the same positions, a configuration reported as one of the most common and efficient for gene expression. We explain these results assuming that the presence of adenines in these early positions enhance gene expression. As expected, specific mRNA levels correlated with the intensity of lacZ expression for each variant. However, the induction of lacZ AGA AGA gene in pth cells accumulated peptidyl-tRNA(Arg4) as well as a short 5'-proximal lacZ mRNA fragment suggesting ribosome stalling due to depletion of aminoacylated-tRNA(Arg4).

Figure 1.

Map not to scale of the pKQV4-based constructs used in this work. Upon addition of the gratuitous inducer IPTG the Lac repressor, encoded by lacI^q (open arrow) dissociates from the operator region O_lac (gray box) and transcription initiates at promoter P_tac (bold arrow). Transcription terminates at the transcription terminator T_rrnb (gray box). The transcription initiated at P_tac drives the expression of the lacZ gene (large open arrow) cloned between the EcoRI and HindIII sites (underlined). The used lacZ variants carried different codons in the +2 and +3 positions. Translation initiation occurs, thorough association of the Shine-Dalgarno (SD, bold) sequence in the mRNAs and the ribosomes, at the translation initiation codon ATG. The constructs were selected in transformed cells resistant to ampicillin conferred by the gene bla (open arrow), which encodes β-lactamase. The relative position of the plasmid replication origin (ori) is indicated. The segments in bold indicate other vector DNA sequences.

Figure 2.

The effect of base composition of the codons located at the positions +2 and +3 on the expression of lacZ gene in wild-type and pth strains. Each pair of columns correspond to the rates in P90C and P90C pth(rap) as indicated. Results represent the mean ± SD of at least two, and up to nine independent experiments. The pairs of codons of each of the lacZ variants, one (+2) above the other (+3), are indicated below each pair of columns. (A) identical codon substitutions; (B) mixed codon substitutions. (see Materials and Methods section for details).

Figure 3.

lacZ mRNA detection during the expression of the lacZ variants. Cultures of the strains P90C (A) and P90C pth(rap) (B) transformed with the indicated lacZ +2, +3 variants were induced with 1 mM IPTG for 40 min. Total RNA was extracted, resolved by gel electrophoresis and transferred to nylon membranes. Upper panels: northern blot assays performed with 5′-end labeled oligonucleotide probes to detect the lacZ mRNAs molecules. Heterogeneous lacZ mRNA and the 5′-proximal lacZ mRNA segment are indicated. As a RNA loading control, lower panels, it is shown the ethidium bromide stained rRNAs transferred to the same membrane which was used for the northern blot assay.

Figure 4.

Analysis of β-Gal synthesis, lacZ mRNA levels, pep-tRNA^Arg4 accumulation and cellular growth during the expression of the lacZ AGA₂ AGA₃ variant in pth cells. Cultures of P90C pth(rap) co-transformed with the plasmid construction harboring the lacZ AGA₂ AGA₃ variant and pACYC (vector), pDC952 (a tRNA^Arg4 overproducer) or pGREC (a Pth overproducer) were induced for lacZ expression by the addition of 1 mM IPTG. (A) Analysis of lacZ mRNA. Total RNA was extracted after 40 min of the induction and a northern blot assay was performed with a specific 5′-end lacZ oligonucleotide probe to detect the lacZ mRNAs. (B) Estimation of accumulated pep-tRNA^Arg4. Total RNA was extracted as indicated above. The samples were halved and each aliquot treated (+) or not (−) with a copper salt solution to hydrolyze aminoacyl-tRNA and therefore, unmask the pep-tRNA. A northern blot assay was performed to reveal the accumulated pep-tRNA^Arg4 using a radioactively labeled oligonucleotide probe specific for tRNA^Arg4. (C) Time course of β-Gal activities determined in culture samples drawn at the indicated times after induction. The inset is an enlargement of the β-Gal activity generated by the cells co-transformed with the empty vector. (D) Growth curves measured as optical density of the P90C pth(rap) strain transformed with the indicated overproducing constructs or with the empty vector (pACYC) after induction with IPTG. For experimental details see Material and Methods section.