Fundamental relationship between operon organization and gene expression

Abstract

Half a century has passed since the discovery of operons (groups of genes that are transcribed together as a single mRNA). Despite the importance of operons in bacterial gene networks, the relationship between their organization and gene expression remains poorly understood. Here we show using synthetic operons in Escherichia coli that the expression of a given gene increases with the length of the operon and as its position moves farther from the end of the operon. These findings can be explained by a common mechanism; increasing the distance from the start of a gene to the end of the operon (termed the "transcription distance") provides more time for translation to occur during transcription, resulting in increased expression. We confirmed experimentally that the increased expression is indeed due to increased translation. Furthermore our analysis indicates the translation initiation rate for an mRNA is sixfold greater during transcription than after its release, which amplifies the impact of the transcription distance on gene expression. As a result of these mechanisms, gene expression increases by ∼40% for each 1,000 nucleotides of transcription distance. In summary, we demonstrate that a fundamental relationship exists between gene expression and the number, length, and order of the genes in an operon. This relationship has important implications for understanding the functional basis of genome organization and practical applications for synthetic biology.

Fig. 1.

Linear relationship between transcription distance and gene expression in operons of different length. Each data point is the mean fluorescence of the first gene in each operon ± SEM from a separate culture. In the schematics of the operons, the green box indicates T710RBS7 and the black box indicates st7. (A and B) Two sets of operons with cfp in the first position of the operon. *, the monocistronic CFP fluorescence values are the same in both A and B. (C and D) Plots are the same as above except the two sets of operons have yfp in the first position. ^‡, the monocistronic YFP fluorescence values are the same in both C and D. (E and F) Gene expression at different transcription distances in the lacZ1 (E) and lacZ2 (F) panels.

Fig. 2.

Expression of a gene depends on its position within an operon. Error bars are ± SEM. # indicates the HL strain number. (A and B) Mean CFP and YFP fluorescence in operon pairs. In each pair, the mean was compared using the two-tailed t test with a significance threshold of 0.05. The values in parentheses are the degrees of freedom and the t value, respectively. (C and D) Mean CFP and YFP fluorescence as a function of the transcription distance within operons. Each data point is from a separate culture.

Fig. 3.

Model of operon transcription and translation. (A) Translation during transcription (transcriptional translation) and following mRNA release (posttranscriptional translation). λ₁, λ₂, and λ₃ are the transcription distances for genes 1, 2, and 3, respectively. (B) The contributions of transcriptional, in-transit, and posttranscriptional translation to the total protein in the cell. The estimated in-transit translation assumes the translation and transcription rates in units of codons per second are approximately equal.

Fig. 4.

The effect of gene position on the dynamics of expression. Data are the mean fluorescence ± SEM. (A) The experimental system used to measure the dynamics of gene expression at different positions within the operon following the induction of transcription with IPTG. All genes had the st7 RBS. (B and C) Mean CFP and YFP fluorescence in the first and third positions of a three-gene operon. (D and E) Relative CFP and YFP fluorescence in the first and third positions of the operon (see main text).

Fig. 5.

Increasing transcription distance increases translation. Error bars are ±SEM. *, the means were compared using a two-tailed t test with a significance threshold of 0.05. (A) Schematics of the cfp and cfp-lacZ operons. (B) Normalized CFP fluorescence in the cfp and cfp-lacZ operons. The difference in the expression of the short and long operons was slightly less than previously observed; this difference may be due to the higher cell density required for RNA extractions. (C) The relative concentration of cfp and cfp-lacZ mRNA normalized to the 16S RNA control as determined by quantitative RT-PCR. (D) Northern blots showing full-length cfp and cfp-lacZ mRNA. The contrast of the whole image was altered solely to enable visualization of the full-length cfp and cfp-lacZ mRNA transcripts; it played no role in the analysis. (E) CFP expression for the samples shown in D. (F) Relative concentration of full-length cfp and cfp-lacZ mRNA for the samples shown in D. (G) Decay of full-length cfp and cfp-lacZ mRNA measured by Northern blots following treatment with rifampicin (t = 0). The fits did not include the final time points due to the inaccuracy of the measurements at such low concentrations.

Fig. 6.

Chromosomal gene expression increases with the transcription distance. (A) The arrangement of the genes at the intS and galK sites in the chromosome. The pLlacO-1 promoter is used for all genes. The 5′-UTR, RBS, and the first 11 codons of the T7 10 gene (purple box) are fused to cfp and yfp. (B) The mean CFP and YFP fluorescence for each strain. Strains were measured in triplicate and error bars indicate the SEM.