Colicross a teaching programme simulating the action of bacterial operons

Transcription

1 and ELS EVER Biochemistry and Molecular Biology Education 28 (2) EDUCATON Colicross a teaching programme simulating the action of bacterial operons Alan G. Clark School of Biologiccrl Scicvcc.s. Vic,tor-icr Uiiiiwsrtj~ o/ lwl~~gi~~~i, P.. Bar 6. Vdliiigton, NCW. Zeolcri~rl Receivcd 6 October 1998: accepted 23 April 1999 Abstract A computer program is described which simulates the response of four different types of bacterial operon to the addition of the ligand regulating their expression. The response is recorded as an increase in specific activity of the bacterial culture of an enzyme coded for by the operon under consideration. The results are displayed bol h numerically and graphically. The behaviour of each of 12 different strains of bacteria carrying each operon may be modelled. These 12 strains comprise the wild type and mutants which may be affected in one or all of the regulator gene, the structural gene or its operator or promoter. The response of single strains of bacteria to the addition of ligand may be modelled or the behaviour of hybrid strains formed by the transfer of genetic material from one strain to another can be simulated. The programme challenges students to identify the regulatory mechanism acting in a given operon by analysing the simulated behaviour of the different strains. The programme is available in DOS or Windows 3.1 or 95/97 formats. ( 2 UBMB. Published by Elsevier Science Ltd. All rights reserved. 1. ntroduction The bacterial operon, and its employment in the regulation of bacterial metabolism, is a concept well entrenched in the syllabi of undergraduate biochemistry courses. The mechanisms of, in particular, the lac operon [1,2], but also others such as the trp operon [3-51 and the Ara operon [6] are described in most biochemistry texts. However, many current treatments give no indication as to the logic that was originally employed in the formulation of the operon concept [1,2]. That this concept pre-dated, and indeed led up to, the identification of mrna, the operator, the promoter, regulatory molecules and the other structures invoked in current descriptions is now often given only light emphasis. The first evidence pointing towards this basic paradigm of biochemistry was as much genetic as it was biochemical. The biochemical mechanism now generally accepted resulted from the synthesis of a wide range of genetic observations related to the regulation of a number of metabolic pathways in E. coli [l]. The program described here provides students with the type of observation that gave rise to the operon model, and requires them to work towards formulating and validating a simple operon mechanism that explains the observations. Since the biochemistry teaching group here does not have the facilities to replicate the original experiments, this program, in various stages of evolution, has been employed in undergraduate courses for over 15 years. Students have found exercises based on it to be both challenging and satisfying. They come to a deeper understanding of how the operons of different character behave and in particular, of how combining different genotypes can give rise to quite different phenotypic expression. The programme gives practice in the basic scientific procedures of formulating an hypothesis and designing experiments to test that hypothesis. t is interactive and encourages analytical thinking and systematic analysis of data. For these reasons it has come to be regarded as a valuable teaching tool. Versions of the program are available for Windows 3.1 or Windows 95/97, and also for MS-DOS which make it available for a&/re.s.s: a1an.clark~vuw.ac.n~ (A.G. Clark). This programinc is based upon. hut represents a major inodification and extension of, the programme Operon. originally written for the Commodore 64. which was provided to the author by Prof. D. Rohinson of Queen Elizabeth College. London //$2. ( 2 UBMB. Published by Elsevier Science Ltd. All rights reserved. P: s ()3-3

2 A. G. Clark / Biochemisiry and Molecular Biologl, Education 28 (2) the older machines which are often relegated to teaching functions (Appendix A). 2. The simulation The basic unit in the simulation is a very simple operon consisting of a structural gene, controlled by a promoter and an operator (see Fig. 1). Expression of the structural gene is governed by a regulator protein (RP), coded for by a non-contiguous regulator gene. RP may activate, binding up-stream of the promoter to facilitate binding of RNA polymerase, or it may be inhibitory, binding at the operator and thus impeding transcription of the structural gene. RP is normally able to bind a low molecular weight ligand which, depending on the model chosen, is either essential for the actions of RP, described above, or else it inhibits these functions. Thus, there are four basic models for the operation of the operon. These are embodied in Fig. 1. There may be induction, where the presence of the ligand is necessary for expression of the structural gene. nduction may be mediated either through a positive mechanism (), where the ligand-protein complex (but not RP alone) activates expression by binding upstream of the promoter or it may be negative (), in which case complexing the ligand to the regulator protein causes the inhibitory protein to detach from the operator site. Alternatively, the ligand may cause repression. t may do so either by a positive mechanism (111), where binding of the ligand to the regulator protein causes the latter to detach from the promoter site where previously it activated transcription of the structural gene, or it may be by a negative effect (V), where the ligand-rp complex binds at the operator and suppresses transcription of the gene. This last mechanism is analogous to the action of the Trp regulator protein [3-51, mediating feed-back repression. Note that the Regulator Gene.~-. 1 i 1. ncluction Positive) 11. '1 Rcgulator protein Regulator (activator) protem nduction,/// t A V nactive Activator complex nactive Regulator GOmpleK 111. Repression (Positive) (Neeat ire)..._. Fig. 1. Opcrons modelled in C'olicr-o.s.r. n each operon system. a regulator gene codes foi- a regulator protein. This is indicated as a circular symbol. This may bind at the promoter (P) or operator () of the structural gene. ts ability to do so is afected by complexing with a ligand which caiises the formation of a conformationally altered form ofthe regulator protein. The complex is shown as a rectangular symbol. (1) nduction by a positivc mechanism: thc inducer forms an activated complex with the regulator protein which binds npstream of the promoter to facilitate transcription. (11) nduction by a negative mechanism. The regulator protcin norinally acts to suppress transcription by binding at the opcrator. Formation of a complex with the igdnd reduces its affinity for the operator and permits transcription to take place at a much increased rate. (ll) Repression by il positive mechanism. The regulator protein binds upstream of the promoter and enhances transcription. Formation of a complex with the ligand causes it to dissociate from the DNA and the rate transcription is thereby reduced. (V) Repression by a negative mechanism. The regulator protein does not. by itself. bind to the DNA. On complexing with the ligand. it undergoes a conformational change which enables it to bind at the operator and thus reduces the rate of transcription. Regulator, Gene ~ ~~ p Operator Structural Gene t- i J V, Repression (Negative)

3 218 A.G. Clork i Biocherni.strx and Moleculur Biologv Educrrtioii 28 (2) assignment of numeric codes to the different operons shown in Fig. 1 differs in sequence from the alphabetical assignments used in the programmes themselves. For any of these mechanisms the wild-type as well as 11 mutant systems arc modelled. These mutants may have a non-functional structural gene or they may have a non-functional operator/promoter site to which the RP or its complex with ligand is unable to bind, and is thus unable to exert its normal effect. This type of mutation corresponds to the operator constitutive " mutant in the lac operon and will exhibit cis-dominant behaviour when it leads to unregulated expression of the structural gene. There may also be mutations in the RP gene: either the protein is not produced at all or a mutant form is produced that can carry out its normal function in the absence of ligand but cannot complex with the ligand, so cannot have its function changed. n the case of negative induction, as is seen in the lac operon, this type of mutant S a super-repressor. The various strains may be designated as, for instance, S'O'R' for the wild-type, S'O'R- for a strain in which a non-functional regulator protein is produced or SO'R' for a strain with a defective structural gene, a normal operator and a regulator protein that can bind to DNA, but is unresponsive to the ligand (e.g. the super-repressed variant of the lac opcron). 3. Examples n the first instance, the bchaviour of single, isolated strains of the bacterium may be modelled. n the DOS version of the programme, incubation of the cultures can be timed from 1-2 min whereas in the Windows programmes incubations are for 2 min only. Ligand can be added at any time from 1 to 2 min. The synthesis of protein follows zero order kinetics and the protein, once produced, decays exponentially. The synthesis and decay of protein is modelled by a simple numerical integration involving ten iterative steps for each time interval. The two competing effects generate a stable steady-state in enzyme conccntration. The lability of the protein is exaggerated so that the consequences of ceasing to produce it arc unambiguous. Transcription is not explicitly modelled. t is implicit that turnover of mrna is more rapid than that of protein so that messenger decay does not affect the kinetics of protein decay. The simulation incorporates a random error, giving the appearance of experimentally obtained data. Data are displayed both numerically and graphically and show the specific activity of the enzyme produced at 2 intervals over the selected time period. The output shows results for a control, in which no ligand is added, as well as the experiment in which the effect of added ligand is observed. An example is shown in Fig. 2, in which the effect of adding ligand to a culture of the ENBME CONCENTRATON Tlme Without With [min] Ligand Ligand nduce, added after 7 min Single strain no = 1C t r l 1 2 Fig. 2. Simulation of adding inducer with wild-type Operon. The display is as seen on screen in the Windows version of the programme, with both numerical and graphical displays of data. wild-type bacterium, carrying a positively regulated, inducible operon (), is modelled. More information may be gained from simulated genetic crosses. t is supposed that the genes constituting the operon may be transferred from a donor strain to a recipient to form a heterozygote that is stable for the duration of the experiment. No specific mechanism of transfer is invoked. However, it is a necessary characteristic of the system that the donor genes are only expressed after transfer to the recipient. For any one of the models chosen, any of the twelve variant operons may be crossed with any other - including self-crosses. A necessary constraint is that the transferred genes are expressed at different times. Genes belonging to the recipient organism will be expressed from the beginning of the experiment. The donor structural gene, coding for the enzyme being measured, will be expressed at 75 min after the start of genetic transfer and the donor RP gene will be expressed at 9 min. These constraints on timing have been adopted so that effects deriving from the expression of these two genes may be distinguished. Ligand may be added at any time, and students will find that addition of it before the expression of the donor RP may produce results quite different from those seen when it is added after the critical 9 min point (see Fig. 3). t is clear that the detail of the simulation departs from what is actually realistic, but some license is necessary to generate clear results, susceptible to a relatively straightforward interpret' '1 t' ion. Another example may be considered. That chosen is a model of repression, via a negative mechanism (Model V). RP cannot bind to the operator unless it is complexed with the ligand. When the complex is bound to the operator, transcription of the structural gene is prevented. n the example shown in Fig. 4A, the donor is the

4 A.G. Clrirk 1 5iochernistr:v and Molecirlol- Biologv Educ~utiorr 28 (2) A 2 1 Minutes 3 t Enz. c 4 [ 3 t 1 [min 1 2 Minutes 3 Fig. 3. Simulation of effects of adding inducer to variants of Operon 11, resulting from the formation of stable heterozygotes from different strains. n A, the donor is the wild type strain and the recipient an RPstrain. n B, the results of the reciprocal cross are presented. The inducer is added () aftcr YO min (see text). Only the graphical portion of the display is shown as seen in the DOS-based version of the program. wild-type whereas the recipient has a defective operator which cannot bind the complex. At the beginning of the experiment the structural gene is being expressed to give a specific activity of 2 units/mg. Addition of repressor at 2 min has no effect. When the donor structural gene is expressed, at 75 min, in the absence of repressor, the specific activity increases to 4 units/mg. No increase occurs in the case where repressor had been added at 2min because the newly transferred structural gene is controlled by an effective operator. For the reciprocal cross, (Fig. 4B) in which the recipient is the wild-type strain, activity at the start of the experiment is again represented as being at a level of 2units/mg. When repressor is added at 2min the activity immediately declines until the donor structural gene is expressed. Since this is not under the control of an effective operator, the activity increases by an additional 2units/mg regardless of the presence or absence of repressor. Fig. 4. Simulation of effects of adding ligand to variants of operon 1V. in which repression proceeds via ii negativc mechanism. n A is shown the result of donating the wild-type operon to an operator constitutive mutant. in which the regulator protein cannot bind to the DNA. Repressor is added at 2 min. n B, the effects of the reciprocal cross ai-e shown. The graphical portion of the Windows display is shown. n the final example, the effect of timing on the experiment is illustrated. The operon is an inducible one which acts by a positive mechanism ~ the RP-inducer complex binds at the promoter to activate transcription (Model ). n Fig. 5A, the recipient is the wild-type organism; the donor one with an absent regulator protein. t responds properly to inducer added at 2 min and enzyme specific activity starts to increase towards a plateau of 2 u/mg. At 6min, the donor structural gene starts to be expressed and the activity increases again towards a value of 4units/mg. On the other hand, if inducer is not added until 9 min, the biphasic increase in enzyme specific activity is completely masked and information is lost. 2

5 22 A.G. Clurk J Biocheniistry and Molecular Bioloa Education 28 (2) Enzyme Activity (Ulmgl 2 P 1 2 lmfn Fig. 5. The effect of time of addition of ligand. n both A and B, the system under scrutiny is that obtained by transferring the operon from the RP- mutant ofinducible operon 11 to a wild-type recipient. Ligand is added at 2 min in A and at 9 min in B. Display as in Fig. 4. Acknowledgements The author is grateful to Dr G.K.Chambers and Dr A.P. Dowsett for feedback on the utility of various versions of this programme and to Dr G.K.Rickards for critical reading of this manuscript. Appendix A window under Windows 3.1 or 95. t was designed to run on machines of the less powerful type (886 processor upwards) commonly found in teaching laboratories. An.ini file can be adjusted to allow for different display modes. n its present form the program requires VGA display or better. The default screen mode is 12 with an option for monochrome display in mode 11. Branching to key points in the programme is effected with the function keys. A Help routine, called by the function key F2, is incorporated into the program. f operating in DOS, screen contents can be dumped to the printer directly provided that a suitable graphics driver has been loaded. The Windows versions are written in Visual Basic 4.. They differ slightly in detail from the DOS version but not in the general principles of operation. They are faster and clearer in presentation than the DOS-based program. The graphics display from these programs may be printed by copying the display to the clipboard and printing from a suitable graphics package. A detailed Help file accompanies the Windows versions. All versions of the program may be downloaded from the URL http: jjwww:. ~u~.ac.iizj - clar.kag/teaching. They are held as self-extracting exe files. The list of references is representative of early developments in the field and may be provided as a resource for students to illustrate the experimental reality underlying the simulations. References [l] F. Jacob, J. Monod, Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3 (1961) [2] B. Muller-Hill. The lac operon. A short history of a genetic paradigm. De Gruyter. Berlin [3] D. McGoegh. J. McGeogli. D. Morse. Synthesis of tryptophan operon RNA in a cell free system. Nature New Biology 245 (1973) [4] J.K. Rose, C.L. Squires. Yanofsky. C Regulation of the irr riwo transcription of the tryptophan operon by purilied RNA polymerase in the presence of partially purified repressor and tryptophan, Nature New Biology 245 (1973) [5] Y. Shimizu. N. Shimizu. M. Hayashi. 117 ritr.o repression of transcription of the tryptophan operon by Trp repressor. Proc. Natl. had. Sci. U.S.A. 7 (1973) [6] E. Engelberg. J. rr. J. Power. N. Lee. Positive control or enzyme synthesis by gene C in the L-arnbinose system. J. Bacteriol 99 (1965) The source code of the.exe file for the DOS version is written in Turbo Basic. t will run from DOS or in a DOS