Profiling of gene-dependent translational progress in cell-free protein synthesis by real-space imaging.
In general, gene-dependent translational progress affects the efficiency of protein expression. To evaluate the translational progress of protein synthesis, it is necessary to trace the time course of translation as well as the quantity of products. Here we present a new method for tracking translation steps in cell-free protein synthesis using atomic force microscopy (AFM). The cell-free protein synthesis system is useful to track the inherent translational progress of a target gene, whereas conventional UV absorption measurement coupled with density gradient fractionation is difficult to analyze such small sample quantities. Because the high resolution of AFM enables us to clearly count the number of ribosomes included in polysomes, polysome profiles can be obtained directly without complicated fractionation. With this method, we could elucidate the detailed polysome profile with only 1 microl of sample solution. We observed the translational progress of green fluorescent protein synthesis, a model of high-expression protein, as well as human retinoid X receptor. Detailed polysome profiles showed different patterns of translational progress and were clearly associated with the results of time-dependent protein expression. Our study suggests the possibility for comprehensive character analysis of inherent gene-dependent translational progress.
Mikamo-Satoh
Eriko
E
Department of Pharmacy, Hyogo University of Health Sciences, Kobe, Hyogo 650-8530, Japan. er_satoh@huhs.ac.jp
Takagi
Akihiko
A
Tanaka
Hiroyuki
H
Matsumoto
Takuya
T
Nishihara
Tsutomu
T
Kawai
Tomoji
T
D002474:Cell-Free System / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018625:Microscopy, Atomic Force; D010442:Peptide Chain Initiation, Translational; D010443:Peptide Chain Termination, Translational; D010957:Plasmids / Q000235:genetics; D011132:Polyribosomes / Q000378:metabolism / Q000648:ultrastructure; D014176:Protein Biosynthesis; D046188:Protein Modification, Translational; D012333:RNA, Messenger / Q000378:metabolism; D047488:Retinoid X Receptors / Q000378:metabolism / Q000648:ultrastructure; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure; D013997:Time Factors; D014169:Transformation, Bacterial
D012333:RNA, Messenger; D047488:Retinoid X Receptors; D049452:Green Fluorescent Proteins