Transcription and translation initiation frequencies of the Escherichia coli lac operon

Kennell, David; Riezman, Howard

doi:10.1016/0022-2836(77)90279-0

Cited by 309 publications

(223 citation statements)

References 42 publications

Supporting

Mentioning

198

Contrasting

Unclassified

Order By: Relevance

“…1B) Met IC--Ilx ----+-- [23] and car [26] maximize hybridization efficiency. In the case of lac, the estimate concerns the lacZ gene only and has been corrected for intrinsic hybridization efficiency [24]. The number of tryptophan synthetase molecules per cell is from [22].…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Synthesis of Escherichia coli carbomoylphosphate synthetase initiates at a UUG codon

Weyens

Rose²,

Falmagne

et al. 1985

European Journal of Biochemistry

View full text Add to dashboard Cite

The ribosome binding region of the messenger RNA for the Escherichia coli carA gene contains two adjacent putative translational start codons, UUG and AUU, both of them unusual. By Edman degradation and mass spectrometry of purified carA protein, we show that only UUG is used in vivo. Translation initiation at UUG in carA appears about half as efficient as at AUG in lacZ.Escherichia coli carbamoylphosphate synthetase consists of two subunits, the products of the genes carA and carB which constitute an operon oriented from A to B [l, 21.The putative translational start codon of carA appears to be unusual; indeed the ribosome binding region of carA messenger RNA presents a Shine-Dalgarno [3] sequence (bold-free type) followed at five nucleotides by the tandem GGAGGGUGUUUUGAUUAAGUCA. Both UUG and AUU have been reported to serve as initiating triplets in E. coli but only in exceptional cases [6 -111.The aim of the present study was to determine whether only one or possibly both of these triplets were used in vivo to initiate carA translation. By means of mass spectrometry and automated Edman degradation, we show that only UUG is used and discuss the implications of these results. MATERIALS AND METHODS Escherichia coli strain UUG AUU [4,5]: S'AUAAAGUGAGUGAAUAUUCUCU-The strain Jef8 (HJL m e t 3 thr carB8) containing plasmid pMC5O has been described [4]. Growth conditionsCultures were grown in minimal medium 132 [12].Enzyme assays were assayed as described previously [l, 131. Carbamoylphosphate synthetase and the carA subunit Enzyme pur ficationNative carbamoylphosphate synthetase was purified as described [14], with the following modifications, from a frozen Ahhreviations. GC/MS, gas-liquid chromatography/mass spectrometry; Pth-, phenylthiohydantoin.Enzyme. Carbamoylphosphate synthetase (EC 6.3.5.5).paste ( z 150 g) of E. coli Jef8 strain containing multicopy plasmid pMC50, grown on minimal medium. All steps were carried out at 4 "C. After the protamine sulfate precipitation (150 mg/g protein) 1 pg RNase and 1 pg DNase were added per ml supernatant. Ammonium sulfate was added to 65% saturation; the precipitate was collected by centrifugation (10000 x g, 15 min) and resuspended in 0.2 M potassium phosphate buffer (pH 7.9) containing 0.5 mM EDTA and 30 mM glutamine supplemented with ammonium sulfate to 65% saturation. The suspension was supplemented with 300 g/l Celite 545 and applied to a column (5 x 40 cm) of Celite 545 equilibrated with the same 65% saturated potassium phosphate buffer. Active fractions were eluted with a linear ammonium sulfate gradient (65 -20%), pooled, precipitated by ammonium sulfate at 65% saturation and centrifuged (10000 xg, 15 min). After solubilization in and dialysis against 0.1 M potassium phosphate buffer (pH 6.8) containing 0.5 mM EDTA and 30 mM glutamine, the preparation was applied to a DEAE-Sepharose CL-6B column ( 5 x 40 cm) containing 200 ml of the agarose gel and washed with the same buffer until the non-absorbed proteins were eluted. Further elution was carried out with a line...

show abstract

Section: Resultsmentioning

confidence: 99%

“…The number of tryptophan synthetase molecules per cell is from [22]. The number of P-galactosidase monomers per cell was calculated from [24] ( …”

Section: Resultsmentioning

confidence: 99%

Synthesis of Escherichia coli carbomoylphosphate synthetase initiates at a UUG codon

Weyens

Rose²,

Falmagne

et al. 1985

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The sub-millisecond misfolded species would not interfere with folding during synthesis because it would appear and dissipate far faster than the rate of synthesis on a ribosome at 10-20 amino acids per second 52,53 or the subsequent folding reactions. Its conservation might reflect the (βα) n -repeat architecture of these barrels and, thereby, the opportunity for local and rapid folding reactions to forms of sufficient stability to be transiently populated during folding.…”

Section: Why Are the On-and Off-pathway Intermediates For (βα) 8 Barrmentioning

confidence: 99%

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

Rao

Forsyth

et al. 2007

Journal of Molecular Biology

View full text Add to dashboard Cite

The structures of partially-folded states appearing during the folding of a (βα) 8 TIM barrel protein, the indole-3-glycerol phosphate synthase from S. solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō-model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (βα) 4 region, modest protection in the neighboring (βα) 1-3 and (βα) 5 β 6 segments and no significant protection in the remaining N-and Cterminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (βα) 2-5 β 6 region after 5 s of folding demonstrates that these species represent kinetically-distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C α Gō-model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of structure offering protection against exchange in the on-pathway intermediate (s). Because the native-centric Gō-model simulations do not explicitly include sequencespecific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō-model simulation.

show abstract

“…The basic transcription rate is set to k t = 0.01 causing the expected time for an RNAp to bind to the promoter to be 1 kt×RN Ap = 2.5 [23]. Reaction rates k tr = 0.00042 and d rbs = 0.01 are set to match the average observed number of proteins produced per RNA E. coli, i.e., 4.2 [51].…”

Section: Toggle Switchmentioning

confidence: 99%

Tuning cell differentiation patterns and single cell dynamics by regulating proteins’ functionalities in a toggle switch

Dai¹,

Healy²,

Yli‐Harja³

et al. 2009

Journal of Theoretical Biology

View full text Add to dashboard Cite

We investigate how the regulation of protein multi-functionalities affect the dynamics of a stochastic model of a toggle switch and the differentiation pattern of cell population regulated by the switch. We study the effects of loss of functionality in DNA-binding and repression and the involvement in differentiation pathway choice. First is shown how the patterns of cell differentiation differ, when each of these functionalities is fully non-functional. Next, tuning the fraction of non-functional proteins regarding the ability to bind DNA is shown to allow fine tuning of the switch and cell differentiation pattern dynamics. Finally, biasing the probability of functionality of the two proteins biases the dynamics of the switch and cell differentiation patterns, especially when transcription factors retain the ability to bind DNA but have lost the ability to repress gene expression. Our results suggest that, besides transcriptional and translational levels of regulation, activation of functionalities in multi-functional proteins are an important regulator of gene networks.

show abstract

Transcription and translation initiation frequencies of the Escherichia coli lac operon

Cited by 309 publications

References 42 publications

Synthesis of Escherichia coli carbomoylphosphate synthetase initiates at a UUG codon

Synthesis of Escherichia coli carbomoylphosphate synthetase initiates at a UUG codon

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

Tuning cell differentiation patterns and single cell dynamics by regulating proteins’ functionalities in a toggle switch

Contact Info

Product

Resources

About