Gene expression simulation:

We have simulated gene expression kinetics to deduce the changes in the rate of translation. Gene expression (DNA to protein) was simulated as a series of mass-action chemical reactions, using Kinetikit/GENESIS (1). Transcription and translation were modeled as rate-limiting forward reaction steps (input rate) followed by a delay line to represent the synthesis. The number of bins in the delay line was equivalent to the number of bases of the mRNA sequence. The steps of gene expression include: transcription initiation and elongation by the RNA polymerase motor (equations a & b), translation initiation and elongation by the ribosome motor (equations c to f), and bioluminescence reaction of the translated luciferase proteins (equations g to m). In the following set of reactions kf and kb are the forward and backward reaction rates and the dissociation constant Kd = kb/kf. Rate constants kf and kb are expressed in time units of seconds and, where applicable, concentration units of M. The whole simulation can be divided into three modules transcription, translation and luminescence.

Transcription  Translation  Luminescence

Transcription:

Pr  RNAP  TrRNAP, (kf = 182 M-1.sec-1) (2) (a)

TrRNAP  N  Pr  RBS, (kf = 4.510-6M-1.sec-1) (Promega kit)(b)

RBS  (Delay line) mRNA  RNAP (input rate = 0.0002/sec,

delay duration = 425sec, delay time step = 0.25sec).(Promega kit)(c)

Translation:

mRNA  A_subunit  A_mRNA, (kf =100 M-1.sec-1, kb =2.25 sec-1)(3) (d)

A_mRNA B_subunit  A_B_mRNA (kf = 0.14M-1.sec-1, kb =2.25sec-1) (e)

A_B_mRNA  (Delay line) mRNA A_subunit  B_subunit Protein

(input rate = 0.106/sec, delay duration = 120sec, delay time step = 0.067sec)(f)

Luminescence:

Protein + S1 Protein_S1 (kf =210-2M-1.sec-1, kb=6 sec-1) (4) (g)

Protein + S2 Protein_S2 (kf =1M-1.sec-1, kb = 10.sec-1) (4) (h)

Protein_S1+S2 Protein_S1_ S2 (kf =1M-1.sec-1, kb = 10sec-1) (4) (i)

Protein_S2+S1 Protein_S1_ S2 ((kf=210-2M-1.sec-1, kb=6sec-1) (4) (j)

Protein_S1_ S2 Protein_P1 + AMP +PPi (kf =30 sec-1)(4) (k)

Protein_P1  Protein_P + h (kf =10sec-1) (4) (l)

Protein_P  Protein + P (kf =0.1sec-1, kb = 1M-1.sec-1) (4) (m)

Photon count = 3.71010 h (n)

In the above reactions, Pr represents promoter sequence, and RNAP is RNA Polymerase. RNAP binds to Pr and forms the open complex (TrRNAP) that is ready for transcription elongation. The formation of intermediate closed complex, which subsequently forms TrRNAP, is not accounted for in the simulation. N represents ribonucleoside triphosphates (rNTPs) that are polymerized by TrRNAP into the mRNA. An intermediate product, ribosomal binding site (RBS), a sequence on mRNA, is synthesized before the formation of mRNA. A_subunit and B_subunit represent the large and small subunits of ribosome respectively. Both subunits subsequently bind to mRNA. A_B_mRNA represents the tri complex of A_subunit, B_subunit and mRNA. S1 represents ATP and S2 luciferin. Protein_P1 is an intermediate product called luciferyladenylate, oxidation of which by atmospheric oxygen gives rise to protein-hydroluciferin complex (Protein_P) and a photon (h). Subsequent dissociation of Protein_P generates protein and hydroxyluciferin (P). Photons generated above are counted by the PMT to measure luciferase activity in real time. Equation (n) shows the scaling factor that is included in the simulation in order to compare experiment and simulation results. We scale the simulation result to account for the quantum efficiency of the setup.

References:

1. Bhalla, U.S. (2003) Prog Biophys Mol Biol81(1): 45-65.

2.Bandwar, R. P., Jia, Y., Stano, N. M. & Patel, S. S. (2002) Biochemistry41, 3586-95.

3.Kierzek, A. M., Zaim, J. & Zielenkiewicz, P. (2001) J Biol Chem276, 8165-72.

4.Brovko Yu. L, O. A. G. m., T.E.Polenova and N.N Ugarova (1994) Biochemistery (Moscow)59, 195-201.