Transcription factors regulate gene expression through their binding to DNA. In a living Escheric... more Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends approximately 90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.
D-dimer testing is widely used as part of the diagnostic algorithm for the exclusion of deep vein... more D-dimer testing is widely used as part of the diagnostic algorithm for the exclusion of deep vein thrombosis (DVT) but is considered of limited in value for ruling DVT in. Since D-dimers are poorly defined, there is no standardization of the assays and this makes reliable comparisons between clinical studies difficult. We report on a performance evaluation of a new marker of activated coagulation (Activated Protein-C in complex with Protein-C inhibitor, APC-PCI complex) compared to two quantitative D-dimer assays (Vidas D-dimer Exclusion and Autodimer). The post-hoc comparison was made on 350 frozen plasma samples from consecutive outpatients suspected of DVT in a multicenter management study including clinical probability score, D-dimer testing, venous ultrasound and contrast venography as part of the diagnostic algorithm. The APC-PCI complex performed inferior to the D-dimer assays in terms of sensitivity: 74 vs. >93%, negative predictive value: 91 vs. >96% and area under the curve: 0.82 vs. 0.9, but showed a significantly higher specificity: 80 vs. 40-60%. Specificity for the APC-PCI complex did not decrease with higher clinical probability score and the positive predictive value was significantly higher than that of the D-dimer assays in the intermediate/high probability cohort (66 vs. <52%). In this probability cohort, high levels of the APC-PCI complex and to a lesser extent, D-dimers, can give positive predictive values of >90% in up to 20% of the patients which indicates important clinical implications. However, for the exclusion of DVT at the pre-specified cut-off level, the APC-PCI complex perform inferior to the D-dimer assays in this study.
Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. ... more Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. It is commonly assumed that the level of repression is determined solely by the equilibrium binding of a repressor to its operator. However, this assumption has not been possible to test in living cells. Here we have developed a single-molecule chase assay to measure how long an individual transcription factor molecule remains bound at a specific chromosomal operator site. We find that the lac repressor dimer stays bound on average 5 min at the native lac operator in Escherichia coli and that a stronger operator results in a slower dissociation rate but a similar association rate. Our findings do not support the simple equilibrium model. The discrepancy with this model can, for example, be accounted for by considering that transcription initiation drives the system out of equilibrium. Such effects need to be considered when predicting gene activity from transcription factor binding strengths.
Negative autoregulation, where a transcription factor regulates its own expression by preventing ... more Negative autoregulation, where a transcription factor regulates its own expression by preventing transcription, is commonly used to suppress fluctuations in gene expression. Recent single molecule in vivo imaging has shown that it takes significant time for a transcription factor molecule to bind its chromosomal binding site. Given the slow association kinetics, transcription factor mediated feedback cannot at the same time be fast and strong. Here we show that with a limited association rate follows an optimal transcription factor binding strength where noise is maximally suppressed. At the optimal binding strength the binding site is free a fixed fraction of the time independent of the transcription factor concentration. One consequence is that high-copy number transcription factors should bind weakly to their operators, which is observed for transcription factors in Escherichia coli. The results demonstrate that a binding site's strength may be uncorrelated to its functional importance.
During mRNA translation, synonymous codons for one amino acid are often read by different isoacce... more During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNAGAGLeu. As predicted, the charged level of tRNAGAGLeu increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNAUAGLeu increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNAGAGLeu and tRNAUAGLeu. Subsequently, we demonstrated that the tRNAGAGLeu codon CUU is, in fact, read also by tRNAUAGLeu, due to a uridine-5-oxyacetic acid modification.
Transcription factors regulate gene expression through their binding to DNA. In a living Escheric... more Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends approximately 90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.
D-dimer testing is widely used as part of the diagnostic algorithm for the exclusion of deep vein... more D-dimer testing is widely used as part of the diagnostic algorithm for the exclusion of deep vein thrombosis (DVT) but is considered of limited in value for ruling DVT in. Since D-dimers are poorly defined, there is no standardization of the assays and this makes reliable comparisons between clinical studies difficult. We report on a performance evaluation of a new marker of activated coagulation (Activated Protein-C in complex with Protein-C inhibitor, APC-PCI complex) compared to two quantitative D-dimer assays (Vidas D-dimer Exclusion and Autodimer). The post-hoc comparison was made on 350 frozen plasma samples from consecutive outpatients suspected of DVT in a multicenter management study including clinical probability score, D-dimer testing, venous ultrasound and contrast venography as part of the diagnostic algorithm. The APC-PCI complex performed inferior to the D-dimer assays in terms of sensitivity: 74 vs. >93%, negative predictive value: 91 vs. >96% and area under the curve: 0.82 vs. 0.9, but showed a significantly higher specificity: 80 vs. 40-60%. Specificity for the APC-PCI complex did not decrease with higher clinical probability score and the positive predictive value was significantly higher than that of the D-dimer assays in the intermediate/high probability cohort (66 vs. <52%). In this probability cohort, high levels of the APC-PCI complex and to a lesser extent, D-dimers, can give positive predictive values of >90% in up to 20% of the patients which indicates important clinical implications. However, for the exclusion of DVT at the pre-specified cut-off level, the APC-PCI complex perform inferior to the D-dimer assays in this study.
Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. ... more Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. It is commonly assumed that the level of repression is determined solely by the equilibrium binding of a repressor to its operator. However, this assumption has not been possible to test in living cells. Here we have developed a single-molecule chase assay to measure how long an individual transcription factor molecule remains bound at a specific chromosomal operator site. We find that the lac repressor dimer stays bound on average 5 min at the native lac operator in Escherichia coli and that a stronger operator results in a slower dissociation rate but a similar association rate. Our findings do not support the simple equilibrium model. The discrepancy with this model can, for example, be accounted for by considering that transcription initiation drives the system out of equilibrium. Such effects need to be considered when predicting gene activity from transcription factor binding strengths.
Negative autoregulation, where a transcription factor regulates its own expression by preventing ... more Negative autoregulation, where a transcription factor regulates its own expression by preventing transcription, is commonly used to suppress fluctuations in gene expression. Recent single molecule in vivo imaging has shown that it takes significant time for a transcription factor molecule to bind its chromosomal binding site. Given the slow association kinetics, transcription factor mediated feedback cannot at the same time be fast and strong. Here we show that with a limited association rate follows an optimal transcription factor binding strength where noise is maximally suppressed. At the optimal binding strength the binding site is free a fixed fraction of the time independent of the transcription factor concentration. One consequence is that high-copy number transcription factors should bind weakly to their operators, which is observed for transcription factors in Escherichia coli. The results demonstrate that a binding site's strength may be uncorrelated to its functional importance.
During mRNA translation, synonymous codons for one amino acid are often read by different isoacce... more During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNAGAGLeu. As predicted, the charged level of tRNAGAGLeu increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNAUAGLeu increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNAGAGLeu and tRNAUAGLeu. Subsequently, we demonstrated that the tRNAGAGLeu codon CUU is, in fact, read also by tRNAUAGLeu, due to a uridine-5-oxyacetic acid modification.
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Papers by Johan Elf