Experimental and computational analysis of translation products in apomyoglobin expression.
This work focuses on the experimental analysis of the time-course of protein expression in a cell-free system, in conjunction with the development of a computational model, denoted as progressive chain buildup (PCB), able to simulate translation kinetics and product formation as a function of starting reactant concentrations. Translation of the gene encoding the apomyoglobin (apoMb) model protein was monitored in an Escherichia coli cell-free system under different experimental conditions. Experimentally observed protein expression yields, product accumulation time-course and expression completion times match with the predictions by the PCB model. This algorithm regards elementary single-residue elongations as apparent second-order events and it accounts for aminoacyl-tRNA regeneration during translation. We have used this computational approach to model full-length protein expression and to explore the kinetic behavior of incomplete chains generated during protein biosynthesis. Most of the observed incomplete chains are non-obligatory dead-end species, in that their formation is not mandatory for full-length protein expression, and that they are unable to convert to the expected final translation product. These truncated polypeptides do not arise from post-translational degradation of full-length protein, but from a distinct subpopulation of chains which expresses intrinsically more slowly than the population leading to full-length product. The PCB model is a valuable tool to predict full-length and incomplete chain populations and formulate experimentally testable hypotheses on their origin. PCB simulations are applicable to E.coli cell-free expression systems (both in batch and dialysis mode) under the control of T7 RNA polymerase and to other environments where transcription and translation can be regarded as kinetically decoupled.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.
Lisa M
LM
Jungbauer
Courtney K
CK
Bakke
Silvia
S
Cavagnero
D000465:Algorithms; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D003062:Codon; D003198:Computer Simulation; D008957:Models, Genetic; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D000596:Amino Acids; D001059:Apoproteins; D003062:Codon; D009211:Myoglobin; D010455:Peptides; D012333:RNA, Messenger; C010857:apomyoglobin