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Key words:transcription, initiation,elongation, termination, nus-factor, RNApolymerase

Abbreviations:RNAP, RNA polymerase;Nus, N-utilization substance; E. coli,Escherichia coli; NTD, amino-terminaldomain; CTD, carboxy-terminaldomain; AR, acidic repeat;EC, elongation complex;ChIP, chromatin immunoprecipitation

Submitted: 02/03/11

Revised: 03/31/11

Accepted: 03/31/11

DOI: 10.4161/trns.2.3.15671

*Correspondence to: Bjrn Burmann;

Email: bjoern.burmann@unibas.ch

Bacterial transcription mediated byRNA polymerase (RNAP) is a highlyregulated process and RNAP action is

modulated during the dif ferent phases ofinitiation, elongation and termination byproteins such as the Escherichia coliNustranscription-factors. Here we discuss thestructural interplay and the mechanisticrole of the Nus-factors that are directlyinvolved in the processivity of elonga-tion, transcription:translation couplingand termination, as well as the varyingeffects of these proteins on transcriptionunder the influence of additional signals.

The Nus transcription-factors wereoriginally identified as part of the E. coliphage N-protein-controlled antitermi-nation system; hence, they were termedN-utilization substances.1 NusA, B, Eand G are important for different levelsof regulation within transcription andtranscription:translation coupling. Theirregulatory effect is often antithetic anddepends on different external signals.

NusA

NusA is a highly conserved elongationfactor identified in bacteria and archaea,2which in E. coli consists of 495 aminoacids (55 kDa) arranged in 6 domains(Fig. 1A). The amino-terminal domain(NTD; amino-acids 1137) interacts withthe RNAP close to the RNA exit chan-nel.3 NusA-NTD is linked via a flexiblehelix to three RNA-binding subdomains,S1 (138201), KH1 (202276) and KH2(277344), forming the central SKK

The role ofE. coli

Nus-factors in transcription regulationand transcription:translation coupling

From structure to mechanism

Bjrn M. Burmann,* and Paul RschLehrstuhl Biopolymere und Forschungszentrum f r Bio-Makromolekle; Universitt Bayreuth; Bayreuth, GermanyCurrent address: Biozentrum; University of Basel ; Basel, Switzerland

domain that binds single stranded RNAof the nascent transcript.4,5 C-terminalto this SKK domain follow the so-calledacidic repeats 1 and 2 [AR1 (345426),

AR2 (427495)], which so far could onlybe identified in E. coli.6Although the lat-ter domains share the same structuraltopology, they exhibit different functions.The only role for AR1 identified thus farin vitro is binding the protein N7 andthe N:AR1 complex may well be partof the antitermination complex in vivo;nevertheless, this could not be shownso far. AR2 has at least two distinct butinterrelated functions: It masks the cen-

tral SKK domain of NusA by forming anintramolecular interdomain complex, thusautoinhibiting RNA interaction of thisdomain;8,9AR2 forms a complex with theC-terminal domain CTD of the-subunitof RNAP (CTD), which supposedlyhinders this regulatory RNAP subunitfrom re-attaching to the UP-element(upstream promoter).8 Primarily, NusAsupports intrinsic termination due to itsinteraction with the hairpin structures ofpause and termination signals3,10 and itmodulates p-dependent termination.11,12

In concert with the other Nus-factors,NusA enables the formation of stable ECs(elongation complexes), which leads toprocessive transcription and read-throughof termination sites.13

NusB

NusB (15.7 kDa) mainly acts as an aux-iliary protein within transcriptional regu-lation, which consists of an all-helical

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NusG

NusG is an essential bacterial regulatorof the RNAP with a size of 20.5 kDa.Proteins homologous to NusG can befound in archaea21and eukaryotes.22Withits two domain structure and a flexiblelinker between the transiently interactingdomains,23NusG is able to interact withdifferent partners and therefore is pre-destined to function as a linker protein

(Fig. 1C).24

The NusG-NTD, residues1116, consists of four central -strandsforming an anti-parallel -sheet sur-rounded by three -helices and is linkedto the CTD, residues 123181, whichitself forms a -barrel composed of fiveanti-parallel -strands.24 The NusG-NTD is supposed to directly interactwith the RNAP through a hydrophobicpatch on its surface25and NusG-CTD isable to bind either to NusE20 to enable

helix 1and irregular strand

2bridge the

two helical bundles of NusB and form aprotein interaction surface of ~1,700 2without any evident structural rearrange-ment.17 In this heterodimer, NusE is thefunctionally active partner, which couldbe shown by its N-protein antitermina-tion activity in the absence of NusB.17In addition to its role in transcription,NusE is involved in translation as part ofthe 30S subunit of the ribosome, where

it exhibits virtually the identical structureas in its NusB complex. NusE belongsto those proteins that finish ribosomalassembly.19 Binding to NusB ascertainsa stable fold of NusE,17and NusB is ableto direct the heterodimer to the EC,17,20where it becomes a central part of the EC.

Whether NusB is important for the intro-duction of NusE into the ribosome andhow this is achieved will be questions forfuture studies.

fold with two perpendicular three-helixbundles.14 NusB is able to bind singlestranded RNA.15,16 Additionally, NusBforms a stable complex with NusE(Fig. 1B),15 that, however, leads only toa slight rearrangement of its helix-bun-dles.17This heterodimerization enhancessignificantly the affinity to RNA,16,18because both proteins form an extendedmosaic RNA-binding interface.17Resultsof combined biophysical, mutational and

genetic investigations indicate that thisinteraction is fine-tuned within the func-tional EC.16

NusE

NusE is an 11.7 kDa protein, which dou-bles as ribosomal protein S10 and exhibitsa four-stranded antiparallel -sheet thatis backed by two -helices on one side.17Upon complex formation with NusB,

Figure 1.(A) Crystal-Structure of Thermotoga maritimaNusA and solution NMR-structures of the two AR-domains of E. coliNusA. The NusA-NTD of

T. maritima(green) is linked via a connecting helix (grey) to the central RNA-binding domains S1 (red), KH1 (blue) and KH2 (aquamarine) of NusA (PDB-

ID: 1HH2).4Additionally shown are the solution NMR-structures of AR1 (brown; PDB-ID: 1WCL) and AR2 (yellow; PDB-ID: 1WCH) domains, 6which so far

could only been identified within E. coli. (B) Crystal-Structure of the E. coliNusB:NusE complex. NusE (green) and NusB (purple) form a tight complex

(PDB-ID: 3D3B).17The single sphere within NusE denotes Ser46, which replaces the ribosome binding loop 4667 in the crystallized construct (see

details in reference 17). (C) Solution NMR-Structure of NusG (NusG-NTD; PDB-ID: 2K06; NusG-CTD; PDB-ID: 2JVV). 24

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complex is formed that induces bend-ing and unwinding of the DNA to forma transcription bubble.29 During RNAsynthesis, several transcription regula-tors bind to the RNAP to ensure a stable

and processive EC. NusA is among thefirst proteins to interact with RNAP afterinitiation.30The NusA NTD binds closeto the RNA exit channel of RNAP3 andforms a stable RNAP complex via NusA-

AR2:CTD-RNAP domain interaction.9The AR2:CTD complex in all likeli-hood prevents CTD from reattachingto DNA,8 a process that would lead toa stalled transcription complex. Recent

Elongation Complexes

E. colicore RNAP consists of five subunits(

2, , and ) of roughly 400 kDa

molecular mass (Fig. 2).28 The and

-subunits form the active site, whereasthe NTDs of the two subunits ascertaincorrect RNAP assembly. The subunitsupports this assembly, but is not essen-tial for RNAP functionality.28 To enableDNA binding, a -factor has to associ-ate to form the holo-RNAP, which isable to move along the double-strandedDNA to recognize promoter sequences.28Once bound to these, an open initiation

transcription:translation coupling or tothe -factor20 to support -dependenttermination. NusG versatile role withintranscription regulation is reflected byits ability to modulate the EC in differ-

ent ways: NusG is able to enhance theelongation rate, an effect probably dueto its ability to suppress transcriptionalpausing,26in turn attributable to its pro-motion of forward translocation of theRNAP;27 NusG is also important foractivation of in vivo and in vitro formost -dependent termination events inorder to maintain transcriptional bound-aries within the bacteria l genome.12

Figure 2.The different ECs within a transcription cycle consisting of RNAP (grey), clamp helices (red), 70(yellow), DNA (black), Rho-hexamer (light-

blue), NusA (dark-green), RNA (red), NusG (dark-blue), NusB (purple), NusE (light-green), 50S ribosomal subunit (sand) and 30S ribosomal subunit

(brown).

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on transcriptiondo ribosomes just loadonto RNAP and push it along the DNA,or do ribosomes induce structural changesin the RNAP and thus act antitermina-tion factor-like to prevent transcriptionalpausing, as proposed by Roberts?37Takentogether, the model for the different states

of transcription will undergo significantrefinement as future work addressing theseimportant questions leads to more detailedunderstanding of transcription regulationand transcription:translation coupling.

Acknowledgments

Funding by the Deutsche Forschungge-meinschaft to PR (Ro617/16-1;18-1) isgratefully acknowledged.

References

1. Friedman DI, Baron LS. Genetic characterization

of a bacterial locus involved in the activity of the Nfunction of phage lambda. Virology 1974; 58:141-8.2. Nudler E, Gottesman ME. Transcription termina-

tion and anti-termination in E. coli. Genes Cells2002; 7:755-68.

3. Ha KS, Toulokhonov I, Vassylyev DG, Landick R.The NusA N-terminal domain is necessary and suf-ficient for enhancement of transcriptional pausingvia interaction with the RNA exit channel of RNApolymerase. J Mol Biol 2010.

4. Worbs M, Bourenkov GP, Bartunik HD, HuberR, Wahl MC. An extended RNA binding surfacethrough arrayed S1 and KH domains in tr anscriptionfactor NusA. Mol Cell 2001; 7:1177-89.

5. Prasch S, Jurk M, Washburn RS, Gottesman ME,Whrl BM, Rsch P. RN A-binding specif icity of E.coliNusA. Nucl Acids Res 2009 ; 37:4736-42.

6. Eisenmann A, Schwarz S, Prasch S, Schweimer K,

Rsch P. The E. coliNusA carboxy-terminal domainsare structurally similar and show specific RNA P- andN interaction. Protein Sci 2005 ; 14:2018-29.

7. Prasch S, Schwarz S, Eisenmann A, Whrl BM,Schweimer K, Rsch P. Interaction of the intrinsicallyunstructured phage lambda N protein with E. coliNusA. Biochemistry 2006 ; 45:4542-9.

8. Schweimer K, Prasch S, Santhanam SP, BubunenkoM, Gottesman ME, Rsch P. NusA interactionwith the -subunit of E. coli RNA polymerase isvia the UP-element site and releases autoinhibition.Structure 2011; In press.

9. Mah TF, Kuznedelov K, Mushegian A, SeverinovK, Greenblatt J. The alpha subunit of E. coli RNApolymerase activates RNA binding by NusA. GenesDev 2000; 14:2664 -75.

10. Gusarov I, Nudler E. The mechanism of intrinsic

transcription termination. Mol Cell 1999; 3:495-504.11. Jin DJ, Cashel M, Friedman DI, Nakamu ra Y, Walter

WA, Gross CA. Effects of rifa mpicin resist ant rpoBmutations on antitermination and interaction withnusAin Escherichia coli.J Mol Biol 1988; 204:247-61.

12. Cardinale CJ, Washburn RS, Tadigotla VR, BrownLM, Gottesman ME, Nudler E. Termination factorRho and its cofactors NusA and NusG silence foreignDNA in E. coli.Science 2008; 320:935-8.

13. Gusarov I, Nudler E. Control of intrinsic transcrip-tion termination by N and NusA: The basic mecha-nisms. Cell 2001; 107:437-49.

the required length12for interaction, as needs to bind to single stranded RNAof about 8090 nucleotides in length tobe able to use its inherent ATP hydrolysisto translocate along the RNA,31 and thatthe freed NusG-CTD is able to bind topromote termination. Nevertheless, not

much is known about the structural andfunctional details of the terminationmechanism, the effect of NusG binding,and the process by which NusG-CTDleaves NusE to bind . Presently, a modelfor the termination mechanism proposesthat, upon interaction with RNA andbinding to NusG, uses its intrinsic ATP-dependent helicase-activity to slide alongthe RNA in 3'-direction into the RNAexit channel.31 This triggers significantwidening of the channel, which leads to acollapse of the transcription bubble. This

process is supported by a weakening of theRNA:DNA hybrid due to an adenine-richDNA-stretch and results in the disassem-bly of the whole EC and in the compo-nents being at disposal for a new round oftranscription.

Concluding Remarks

The weakest link in the chain of informa-tion on the transcription process in E. coliat present is the structure of the RNAP

itself. E. coliRNAP could not be crystal-lized yet, probably due to large insertionsin its- and-subunits, which are in closeproximity to important regulatory siteslike the -clamp-helices and the -lid-domain.28Therefore, most of the modelsfor RNAP interactions are based on ahigh-resolution structure from Thermusthermophilus35 and a recently publishedstructural model of E. coli RNAP,36which awaits structural and functionalvalidation. Knowledge about the effectsinduced by proteins like NusG and NusA

that interact directly with RNAP is verylimited. Specially, little is known aboutthe recently identified direct -bindingto RNAP that occurs upon initiation toelongation transition.31 Indeed, in manycases, not even the interacting domainsof RNAP have been clearly identifiedso far. Structural details of the :NusGinteraction and the termination mecha-nism are still not known. Questions alsoremain on the influence of the ribosome

Chromatin Immunoprecipitation (ChIP)-chip analysis30and in vivo assays31showedthat the -termination factor already col-locates with elongating RNAP at thisearly stage and that stays bound to theRNAP throughout the whole transcrip-tion circle,30-32an interaction that is inde-

pendent of the presence of the nascentRNA chain.31 On a structural level,nothing is known yet about the :RNAPinteraction, but the architecture of thiscrucial linkage is part of several ongo-ing research programs. With progressingelongation, the affinity of the -factor toRNAP gradually decreases, accompaniedby displacement of R2 (region 2) fromits binding site at the -clamp-helices ofRNAP by the NTD of NusG.25The for-mation of this -less EC is an importantcheckpoint within the transcription circle.

If no additional factors associate with theEC at this stage, the RNAP is unable toeffectively transcribe the whole DNA,it only produces short abortive RNAs,33and presumably the NusG-CTD is ableto bind that will be loaded onto thenascent RNA and initiate the formation ofa termination complex, leading directly tocomplete complex disassembly. This pre-mature termination is avoided by bindingof the NusB:NusE heterodimer to singlestranded RNA,16-18 an interaction crucial

for processive transcription.

16

This interac-tion allows direct binding between NusEand NusG-CTD and enables coupling ofthe EC to ribosomes.20

The rate of transcription could beshown to be ribosome-dependent as thisrate depends on the translation rate, lead-ing to the model that RNAP waits forthe leading ribosome to be loaded ontoRNAP,34presumably by the NusE:NusGinteraction,20 and a train of ribosomes,the so-called polysome, is able to follow.This flexibility to adjust the transcrip-

tional yield to their translational needs34

is unique to bacteria, as both processes arespatially connected in contrast to the situ-ation in eukaryotic cells, where transcrip-tion and translation progress in differentcellular compartments. At the end of bac-terial operons, ribosomes encounter non-sense codons and the leading ribosome,as well as the ones following, is removedfrom the RNA. This would ascertainthat the mRNA can quickly accumulate

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30. Mooney RA, Davis SE, Peters JM, Rowland JL,Ansa ri AZ, Landick R. Regu lator traf fick ing onbacterial transcription units in vivo. Mol Cell 2009;33:97-108.

31. Epshtein V, Dutta D, Wade J, Nudler E. An al lostericmechanism of rho-dependent transcription termina-tion. Nature 2010; 463:245-9.

32. Peters JM, Mooney RA, Kuan PF, Rowland JL, KelesS, Landick R. R ho directs widespread termination ofintragenic and stable RNA transcription. Proc Natl

Acad Sci USA 2009; 106:15406-11.33. Goldman SR, Ebright RH, Nickels BE. Direct detec-tion of abortive RNA transcripts in vivo. Science2009; 324:927-8.

34. Proshkin S, Rahmouni AR, Mironov A, NudlerE. Cooperation between translating ribosomes andRNA polymerase in t ranscription elongation. Science2010; 328:504-8.

35. Vassylyev DG, Vassylyeva MN, Perederina A, TahirovTH, Artsimovitch I. Structural basis for transcriptionelongation by bacterial RNA polymerase. Nature2007; 448:157-62.

36. Opalka N, Brown J, Lane WJ, Twist KF, Landick R,Asturias FJ, Darst SA. Complete struc tura l modelof Escherichia coli RNA polymerase from a hybridapproach. PLoS Biol 2010; 8 :1000483.

37. Roberts JW. Molecular biology. Syntheses that staytogether. Science 2010; 328:436-7.

22. Hartzog GA, Wada T, Handa H, Winston F. Evidencethat Spt4, Spt5 and Spt6 control transcription elon-gation by RNA polymerase II in Saccharomyces cerevi-siae. Genes Dev 1998; 12:357-69.

23. Burmann BM, Schweimer K, Scheckenhofer U,Rsch P. Domain interactions of the transcription-translation coupling factor E. coliNusG are intermo-lecular and transient. Biochem J 2011; 435:783-9.

24. Mooney RA, Schweimer K, Rsch P, Gottesman ME,Landick R. Two structurally independent domains of

E. coliNusG create regulatory plasticity via distinctinteractions with RNA polymerase and regulators.J Mol Biol 2009; 391:341-58.

25. Belogurov GA, Mooney RA, Svetlov V, Landick R,Artsi movitch I. Functiona l speciali zation of transcrip-tion elongation factors. EMBO J 2009; 28:112-22.

26. Artsimovitch I, Landick R. Pausing by bacterial RNApolymerase is mediated by mechanistically distinctclasses of signals. Proc Natl Acad Sci USA 2000;97:7090-5.

27. Herbert KM, Zhou J, Mooney RA, Porta AL,Landick R, Block SM. E. coliNusG inhibits back-tracking and accelerates pause-free transcriptionby promoting forward translocation of RNA poly-merase. J Mol Biol 2010; 399:17-30.

28. Severinov K. RNA polymerase structure-function:Insights into points of transcriptional regulation.Curr Opin Microbiol 2000; 3:118-25.

29. Mooney R A, Darst SA, Landick R. Sigma and RNApolymerase: An on-again, off-again relationship? MolCell 2005; 20 :335-45.

14. Altieri AS, Mazzulla MJ, Horita DA, Coats RH,Wing fie ld PT, Das A, et al. The struct ure of thetranscriptional antiterminator NusB from Escherichiacoli. Nat Struct Biol 2000; 7:470-4.

15. Mason SW, Li J, Greenblatt J. Direct interactionbetween two Escherichia coli transcription antiter-mination factors, NusB and ribosomal protein S10.

J Mol Biol 1992 ; 223:55-66.16. Burmann BM, Luo X, Wahl MC, Rsch P, Gottesman

ME. Fine tuning of the E. coliNusB:NusE complex

affinity to BoxARNA is required for processive anti-termination. Nucleic Acids Res 2010; 38: 314-26.17. Luo X, Hsiao HH, Bubunenko M, Weber G, Cour t

DL, Gottesman ME, et al. Structural and functionalanalysis of the E. coliNusB-S10 transcription a ntiter-mination complex. Mol Cell 2008; 32:791-802.

18. Greive SJ, Lins AF, von Hippel PH. Assembly of anRNA-protein complex. Binding of NusB and NusE(S10) proteins to boxARNA nucleates the formationof the antitermination complex involved in control-ling rRNA transcription in Escherichia coli. J BiolChem 2005; 280 :36397-408.

19. Noller HF, Nomura M. Ribosomes. In: NeidhardtFC, Curtiss R, Ingraham J, Lin ECC, Low KB,Eds. Escherichia Coli and Salmonella: Cellular andMolecular Biology. Am Soc Microbiol 1996; 1:167-86.

20. Burmann BM, Schweimer K, Luo X, Wahl MC,

Stitt BL, Gottesman ME, Rsch P. A NusE:NusGcomplex links transcription and translation. Science2010; 328:501-4.

21. Kyrpides NC, Ouzounis CA. Transcription inarchaea. Proc Natl Acad S ci USA 1999; 96:8545-50.