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Ribosomal Protein L4 and Transcription Factor Nusa Have Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Ribosomal protein L4 and transcription factor NusA have separable roles in mediating termination lo transcription within the leader of tile S operon of Escherichia coli lanice M. Zengel and Lasse Lindahl Department of Biology, The University of Rochester, Rochester, New York 14627 USA Ribosomal protein L4 of Escherichia coli autogenously regulates both transcription and translation of the 11-gene S10 operon. Transcription regulation occurs by L4-stimulated premature termination at an attenuator hairpin in the SI0 leader. This effect can be reproduced in vitro but depends on the addition of transcription factor NusA. We show that NusA is required to promote RNA polymerase pausing at the termination site; such paused transcription complexes are then stabilized further by r-protein L4. The L4 effect is observed even if the protein is added after the NusA-modified RNA polymerase has already reached the pause site. Genetically separable regions of the S10 leader are required for NusA and IA action: The attenuator hairpin is sufficient for NusA-dependent pausing, but upstream elements are necessary for L4 to prolong the pause. [Key Words: Transcription; termination; r-protein L4; NusA; attenuation] Received August 24, 1992; revised version accepted October 14, 1992. The S 10 operon of Escherichia coli contains the genes for level of translation: Apparently only the S10 operon ex- 11 ribosomal proteins. Like other r-protein operons in E. hibits control of transcription termination. coli, the S10 operon is autogenously regulated by one of We have been focusing on L4-mediated transcription its products, the 50S subunit protein L4 (Lindahl and control to learn more about the general process of termi- Zengel 1979; Yates and Nomura 1980; Zengel et al. nation, as well as the specific mechanism by which an 1980). This protein, which is encoded by the third gene r-protein regulates expression of its own operon. Our ear- of the operon, regulates the operon by two genetically lier experiments showed that, both in vivo and in vitro, distinct mechanisms: It inhibits transcription by causing excess L4 stimulates termination of transcription -140 RNA polymerase to terminate within the S10 leader bases from the start of transcription, within a string of (Lindahl et al. 1983; Freedman et al. 1987; Zengel and U's on the descending side of a stable hairpin structure Lindahl 1990a), and it inhibits translat.ion by preventing {Lindahl et al. 1983; Zengel and Lindahl 1990a, b}. This initiation of translation of the most proximal gene of the process is independent of translation control and re- S10 operon (Yates and Nomura 1980; Freedman et al. quires only sequences contained within the first 150 1987; Lindahl et al. 1989). bases of the S10 leader [Freedman et al. 1987; Zengel and Both regulatory processes are induced when the syn- Lindahl 1990a). Furthermore, in vitro studies {Zengel thesis of r-protein L4 exceeds the synthesis of 23S rRNA. and Lindahl 1990b, 1991) have revealed that L4-mediated Because L4 is a "primary binding protein" that binds attenuation control is dependent on protein NusA, a directly to 23S rRNA in the early steps of 50S assembly transcription factor required for efficient N- and Q-me- (Spillmann et al. 1977), newly synthesized L4 is nor- diated antitermination in bacteriophage h {Friedman and mally rapidly consumed by the ribosome assembly pro- Gottesman 1983; Grayhack et al. 1985). cess. If the protein's target on 23S rRNA becomes limit- Because NusA is known to generally slow the elonga- ing, free L4 accumulates and inhibits expression of its tion rate and to enhance RNA polymerase pausing at own operon. Similar autogenous control mechanisms specific sites in a variety of transcription units (Yager regulate expression of other r-protein operons and and yon Hippel 1987), we wanted to know whether paus- thereby help to maintain balanced expression of r-pro- ing is involved in the L4-mediated attenuation control. tein and rRNA genes (for review, see Lindahl and Zengel Here, we report kinetic studies showing that NusA does 1986; Jinks-Robertson and Nomura 1987). However, induce RNA polymerase to pause within the S IO leader. these other r-protein operons are regulated only at the Unlike other examples of regulation of transcription ter- GENES & DEVELOPMENT6:2655-2662 91992 by Cold Spring Harbor LaboratoryPress ISSN 0890-9369/92 $3.00 2655 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Zengel and Lindahl ruination and antitermination where NusA-enhanced pausing occurs well upstream of the termination site (Landick and Yanofsky 1984; Chan and Landick 1989; Roesser and Yanofsky 1990; Andersen et al. 1991), the pause in the S10 leader is at the site of in vivo L4-stim- ulated termination. L4 greatly prolongs the pause, even if the r-protein is added after RNA polymerase has already reached the site. Furthermore, we show that genetically separable regions of the S 10 leader are required for estab- lishing the NusA-dependent pause and for L4 stabiliza- tion of the paused complex. Results Termination of transcription on plasmids pLL226 and 39B Figure 2. Effect of L4 on in vitro transcription from plasmids The plasmids used to characterize the NusA-dependent, pLL226 and 39B. Single-round transcription reactions (40 ~1) contained NusA, 5 ~Ci of [32p]UTP, the indicated DNA tem- L4-stimulated termination of transcription are pLL226 plate, and 160 nM r-protein L4 ( + L4) or, as a control, r-protein and its derivative pLL226--39B, hereafter called 39B (Zen- $7 [-L4; $7 has no effect on the transcription reaction (Zengel gel and Lindahl 1990a, b). Plasmid pLL226 contains the and Lindahl 1990b}1. After 5 min of incubation at 37~ the S10 operon promoter and the proximal 165 bases of the reactions were terminated and the transcription products were S10 leader upstream of the terminator from the rrnC analyzed on a denaturing urea/polyacrylamide gel (for details, transcription unit {Fig. 1A). Plasmid 39B is a deletion see Materials and methods). Bands corresponding to read- derivative of pLL226 containing the promoter and prox- through {RT) and attenuated [ATT and ATT') RNAs are indicated. imal 149 bases of the leader followed by the rrn C termi- nator (Fig. 1A). The secondary structure of the S10 leader RNA encoded by these plasmids is shown in Figure lB. Previous in vivo studies indicated that the portion of between the two plasmids in the relative amounts of the S10 leader contained on both pLL226 and 39B is suf- attenuated transcripts ending at the ATT and ATT' sites ficient for L4-stimulated termination of transcription (Fig. 1B}. The ATT site [which corresponds to the in vivo (Zengel and Lindahl 1990a). Moreover, in vitro transcrip- end of L4-stimulated attenuated transcripts (Zengel and tion studies showed that for both plasmids the addition Lindahl 1990a)] was preferentially used by RNA poly- of purified L4 protein stimulated transcription termina- merases transcribing plasmid 39B DNA, whereas both tion in the region of in vivo termination (Zengel and sites were used by RNA polymerases transcribing Lindahl 1990b, 1991). Although transcription from both pLL226. Because these two templates have identical S 10 pLL226 and 39B was inhibited to about the same extent operon sequences throughout the transcribed region, by L4 in vivo and in vitro, the experiment shown in these differences probably reflect an effect of the termi- Figure 2 illustrates a subtle but reproducible difference nator-distal DNA sequence on elongation. Downstream Figure 1. The S10 ribosomal protein op- eron. (A) Genetic map of the ll-gene S10 B Attenuator hairpin operon [top). Below are expanded maps of plasmids pLL226 and pLL226-39B carry- AUc G %; ing the indicated regions of the S10 leader ,uA Au, co cloned upstream of the rrnC terminator co (Zengel and Lindahl 1990a,b). The site of co % u u L4-stimulated termination is shown by co uA the arrow labeled att. (B) The secondary r o A A" structure of the S10 leader (Shen et al. A PSIO 1 kid 1oo.I~.,- 2B $10 operon: uA 1988). Sites of in vivo (ATT; Zengel and 50 9 ouuA GC Lindahl 1990a) and in vitro (ATT and AAUU ~ A J% %," ,u ATT'; Zengel and Lindahl 1990b) termina- ou J ~ .~176 ., d]-*- ATT tion are indicated. The 3' ends of the ua AU A U U~u~, uu% u.c 39B,L 9~c co leader carried in deletion derivatives 39B '~s"~,o ~ ' rrnC cm -, ,* ~u 9 t term. ooc 9 C-'MACAAUQcU" UUAUAAAAUAAUU UC .... and 2B are also indicated. The shaded re- pLL226: ...( $10 leader ] U~3= gion shows the portion of the leader de- :=1 ', UA *r 9 9~ .~ leted in mutant A72. The boxed sequence AUr AU~ pLL226-39B: "t$10 leader" ~[ZI- GGAG indicates the Shine-Dalgamo se- rrnC quence of the S10 gene. term. t A72 ~ 2656 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Roles of NusA and 1,4 in termination sequence effects on termination and pausing have also With NusA, pausing was very efficient: Within 30 sec of been reported in other systems (Telesnitsky and Cham- the start of transcription, virtually all of the RNA poly- berlin 1989; Lee et al. 1990; Reynolds and Chamberlin merases were paused at the ATT/ATT' site, whether or 1992). For quantitation of the amount of "attenuated" not L4 was present in the reaction (Fig. 3A, c and d; Fig. transcripts, we pooled the two RNA classes. 3B). In the absence of L4, most RNA polymerases were then released slowly and proceeded quickly to the rrnC terminator (Fig.
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