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Initiation factor activities for natural messenger RNA translation are markedly reduced in extracts from stationary phase Escherichia coli, as compared to growing cells. Immunological techniques demonstrate that the amount of initiation protein IF3 is actually decreased in nongrowing cells. These changes result in a modified mRNA specificity in the stationary cell extracts.Cell-free extracts from ageing bacterial cultures are generally less active in synthesizing proteins than those derived from logarithmic cultures [I -41. This loss of activity is paralleled by a reduction in polysome content with concomitant increase in free ribosomes (mostly 7 0 3 monomers), a situation postulated to reflect defective initiation of the translation process [4,5]. Escherichia coli ribosomes from stationary cells are less active even for the translation of artificial templates such as poly(U) [2], but in a previous study [6] we showed that these ribosomes can be reactivated in witro for poly(U) translation. However, extracts from stationary phase E . coli even when functioning with poly(U), are inactive with natural mRNA unless crude ribosomal-wash factors are added. This result suggested that these extracts were deficient in some of the initiation factors present in this fraction [7] and prompted us t o analyze in more detail the variation in these factors in the different growth phases of E . coli.The present results show that extracts from overgrown cultures of E . coli are deficient in the three initiation factors of protein synthesis. Not only is the initiation activity reduced but the actual amount of initiation protein IF3 is decreased in aged E . coli cells. Moreover, differences in mRNA specificity were found between factors from growing and non-growing cultures, which might reflect an alteration in the balance between different templatespecific initiation factors present in E . coli [S]. ~- Abbreviation. P O I~(~) ,polyuridylic acid. Definition. A,,, unit, the quantity of material contained in I ml of a solution which has an absorbance of 1 a t 260 nm, when measured in a 1-cm pathlength cell. MATERIALS AND METHODS E . coli MRE Preparation of the Ribosomes andCrude Initiation Factors E . coli was grown in carboys a t 37 "C in P broth (containing per liter l o g peptone, 5 g NaCl, 3 g beef extract and 1 g glucose). Log-phase cells were harvested at an absorbance of 80Klett units (about 5 x lo8 cells/ml) ; to obtain stationary phase cells, cultures having reached 200 Klett units were maintained with aeration for another four hours before harvesting. The harvest procedure and preparation of supernatant and active crude ribosomes were as described elsewhere [6], except that cells were broken by alumina grinding. To prepare saltwashed ribosomes and crude initiation factors, crude ribosomes were homogenized overnight in washing buffer ( I M NH,Cl, 0.03 M Tris-HC1 pH 7.5, 0.01 M MgCl,, 14 mM 2-mercaptoethanol) ; this suspension was centrifuged a t 150000 x g for 2.5 h in a Spinco rotor 50-Ti and the resulting pellet homogeni...

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