DIRECTION OF IN VIVO DEGRADATION OF TRYPTOPHAN MESSENGER RNA - A CORRECTION
DIRECTION OF IN VIVO DEGRADATION OF TRYPTOPHAN MESSENGER RNA - A CORRECTION
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DOI：
10.1038/223040a0
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发表时间：
1969-01-01
期刊：
影响因子：
64.8
通讯作者：
YANOFSKY, C
中科院分区：
文献类型：
--
作者：
MORSE, DE;MOSTELLER, R;YANOFSKY, C
8 O 2 4 6 8时间（min）大。L. trp操纵子的转录。一、在去抑制条件下接近稳态; B，顺序转录的单波。0时，IA（20 μ g/ml.）加入到E. coliW 3110（8 × 10 - 6个细胞/ml.）在30 C下剧烈通风生长;在1-5毫升下，L-色氨酸（100 pag/ml）加入到一半培养物中（B）。在指定的时间，10 ml。用 * H-尿苷（20 Ci/mmole，4 μ Ci/ml）脉冲标记传代培养物，并在0-5分钟后通过与12 ml.氯霉素的粉碎的冷冻溶液（0-4 mg/ml），NaN（20 mM）、MgCl（5 mM）和tris Cl缓冲液（20 mM，pH 7-3）。如前所述纯化JNA **，并与从携带不同trp操纵子基因的p80质粒纯化的DNA杂交 ***。用退火到硝酸纤维素滤膜上的DNA进行杂交;细节已在别处描述 **。结果表示为每100 pag RNA杂交的H-RNA的净cpm，减去通过与来自亲本非转导噬菌体的DNA杂交获得的空白 **;在这些实验中，空白值范围为36-95 cpm/100 pag。trp操纵子中的基因顺序是操纵子-EDCBA。图中显示了用于杂交的噬菌体DNA中trp基因的含量，在图2所示的实验中用这种方法检测了trp mRNA的降解过程。在此，在去阻遏和随后的阻遏之后的不同时间将 * H-尿苷加入培养物的部分中。在加入 * H-尿苷1分钟后加入非放射性尿苷的“追踪”，但在稀释细胞内放射性前体方面基本无效;因此观察到在加入追踪后转录的mRNA区域的明显标记（例如，图2A，C的转录，然后BA）。从图2中的标记模式可以清楚地看出，trp操纵子是按顺序转录的，顺序为E和D;科罗; Cast; B和A。因此，在图2A中，我们看到E和D的转录在3分钟内完成，mRNA区域对应于C。和BA仅在稍后的时间被最大程度地标记。当在去阻遏后4-5或5分钟加入 * H-尿苷时，未能检测到标记的E、D和Co. mRNA区域（图1A和1B）。图2C和D）证实了聚合酶分子的波此时已经通过这些区域的结论（比较图1B）。
8 O 2 4 6 8 Time (min) big. l. Transcription of the trp operon. A., Approach to steady state in dcrepression conditions; B, single wave of sequential transcription. At 0-time, IA (20 pug/ml.) was added to a culture of E. coli W3110 (8 x 10" cells/ml.) growing with vigorous aeration at 30 C; at 1-5 miri, L-tryptophan (100 pag/ml.) was added to half of the culture (B). At the indicated times, 10 ml. subcultures were pulse-labelled with* H-uridine (20 Ci/mmole, 4 guCi/ml.), and killed 0-5 min later by rapid mixing with 12 ml. of a crushed, frozen solution of chloramphenicol (0-4 mg/ml.), NaN (20 mM), MgCl,(5 mM), and tris Cl buffer (20 mM, pH 7-3). JNA was purified as described earlier** and hybridized with DNA purified from p80 phages carrying different genes of the trp operon***. Hybridization was done with DNA annealed to nitrocellulose filtersº*; details have been described elsewhere**. Results are presented as the net cpm of" H-RNA hybridized per 100 pag of RNA, after subtraction of a blank obtained by hybridization to DNA from the parental nontransducing phage**; in these experiments the blank values ranged from 36-95 cpm/100 pug. The order of genes in the trp operon is operator-EDCBA. The trp gene content of the phage DNA used for hybridization is indicated in the figure.The course of degradation of trp mRNA was examined in this way in the experiment shown in Fig. 2. Here,* H-uridine was added to portions of a culture at different times following derepression and subsequent repression. A “chase” of non-radioactive uridine was added 1 min after the addition of* H-uridine, but was largely ineffective in diluting the intracellular radioactive precursor; thus appreciable labelling of mRNA regions transcribed after the addition of the chase was observed (for example, Fig. 2A, transcription of C, and then BA). It is clear from the labelling pattern in Fig. 2 that the trp operon was transcribed sequentially, in the order E and D; Coro; Cast; B and A. Thus in Fig. 2A we see that transcription of E and D is completed by 3 min and mRNA regions corresponding to C. and BA are maximally labelled only at later times. Failure to detect labelled E, D and Co. mRNA regions when* H-uridine is added at 4-5 or 5 min after derepression (Figs. 2C and D) substantiates the conclusion that the wavo of polymerase molecules had already passed over these regions by this time (compare Fig. 1B).