Four cold-sensitive mutants of Escherichia coli, which have defects in the maturation of the 50S ribosomal subunit, were isolated. Each of the mutations was shown to map at a different locus. The loci were assigned the name rim (ribosome maturation) and were shown to map as follows: rimA is co-transduced with ilvD and with pyrE; rimB is co-transduced with aroD; conjugation experiments limited rimD to a region between ilv and malB, and conjugation experiments limited rimC to the 22 to 30 min region of the chromosome. In merodiploids heterozygous for rimA, rimB, or rimD, the wild-type allele was shown to be dominant to the mutant allele. The observation that the rim loci lie outside the strA region and separate from each other, as well as the recessive character of the rim loci, suggests that the mutants may be defective in ribosome maturation factors rather than being defective in ribosomal structural proteins.

alpha-Amanitin-resistant clones were selected in the mouse lymphoblastoid cell line L5178Y. One resistant clone, named A169b, was recloned and the properties of its DNA-dependent RNA polymerases were examined. The RNA polymerase II activity from A169b differs from the parental cell line in that approximately half the activity is resistant to 0.5 microgram/mL alpha-amanitin, while the parental enzyme is 50% inhibited at 0.005 microgram/mL. The enzymes from A169b and the parental line were purified free of polymerase III and their properties compared. The two preparations were identical in their apparent affinities for the four nucleoside triphosphates, in their salt and divalent cation preferences, and in their preference for denatured over native DNA. They differed in their response to alpha-amanitin. The apparent K1 for the parental enzyme was 3.5 X 10(-9) M; plots of 1/V vs. alpha-amanitin concentration gave a biphasic curve with A169b enzyme. The two apparent K1 values were 4.1 X 10(-9) and 2.1 X 10(-6) M. In addition, the enzyme from A169b showed a twofold higher activity on poly [d(AT)] as template, compared to native DNA, than that of the parental enzyme. Other template preferences may be affected, but differences were marginal. These results indicate that mutation to alpha-amanitin resistance may alter other enzymatic parameters; such mutations may be helpful in elucidating structure-function relationships in these complex enzymes.

Cells of the cultured hamster cell line V79 were labeled with tritiated adenosine and incubated for up to 30 min in the presence of inhibitors of glycolysis and oxidative phosphorylation. These inhibitors were (a) 5 mM KCN plus 5 mM iodoacetate, (b) 5 mM KCN plus 5 mM KF, and (c) 15 mM KCN plus 15 mM KF. The fate of the tritium label was examined during incubation with inhibitors and also during subsequent incubation in growth medium in the absence of inhibitors. The tritiated ATP pool was found to decrease in cells incubated in the presence of any of the inhibitor combinations, but only in the presence of 15 mM KCN plus 15 mM KF was this pool decreased below the level of detection. After cells were incubated with KCN plus KF, a high level of ATP was recovered when the inhibitors were removed. Cells incubated with KCN plus iodoacetate retained depletion levels of ATP. Plating efficiency and trypan blue staining showed that KCN-KF treated cells retained viability, whereas KCN-iodoacetate treated cells did not. Cells were examined for ability to take up tritiated uridine before, during, and after depletion of ATP by incubation in the presence of 15 mM KCN plus 15 mM KF. These cells were found to have a variation in uridine uptake that was related directly to intracellular ATP level. Cells in which the ATP was very low exhibited little or no uridine uptake, whereas cells in which the ATP level was near normal exhibited normal uridine uptake.

The assembly of E. coli ribosomes has been studied through the analysis of a new group of ribosome maturation mutants. These mutants, all blocked in a late stage in the maturation of 50s ribosomes, map at four different sites on the chromosome. These sites are distant from the known ribosomal protein sites a t the str-aro E region of the chromosome. The ribosome precursor particles of the mutants contain precursor-type 2 3 s RNA (p23 RNA) and 5s RNA. 4 3 s particles of one of the mutants contain all but one of the normal complement of proteins. Precursor 4 3 s particles from this mutant can be converted to particles with sedimentation values around 50s by incubation with extracts from either the wild-type organism or from other mutants. This in vitro conversion process differs considerably from the process of ribosome reconstitution and indicates a role for extrinsic factors in the maturation of E. coli ribosomes. I NTRO DUCT1 ONMany of the important physical-chemical problems that are related t o ribosome function and assembly are being studied by a wide range of technical approaches. In many of these studies the in vitro reconstitution system of Traub and Nomura (1) is an indispensible component. The in vitro reconstitution of 30s ribosomes of Escherichia coli led t o the important notion that ribosomes of bacteria are formed by a self-assembly mechanism. However, this concept may not apply t o the processes which govern ribosome assembly inside the cell.The biosynthesis o f ribosomes in vivo requires the participation o f a large number of gene products: the genes for structural components ( 3 different RNA molecules and 55 different proteins) as well as the genes which specify a variety of RNA nucleotide methylases, RNA trimming enzymes and at least one enzyme that acetylates a protein of 166 0 1974 Alan R.

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