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Annu.Rev. Mic.,obiol. 1993.47:94543
Copyright©1993by AnnualReviewsInc. All rights reserved

THE TN5 TRANSPOSON

Annu. Rev. Microbiol. 1993.47:945-964. Downloaded from arjournals.annualreviews.org
by University of Wisconsin - Madison on 10/19/05. For personal use only.

W. S. Reznikoff
Department
,~)f Biochemistry,Collegeof Agriculturaland Life Sciences,University
of Wisconsin-Madison,

Madison, Wisconsin 53706

KEYWORDS:
transposase, end sequences, gene regulation, cis-activity

CONTENTS
Introduction
...........................................
The Reguhttion of Transposase and Inhibitor Protein Synthesis ............
The Transposase N-Terminal Sequence Is Critical for Sequence-Specific DNA
Binding and Other Transposase Activities
................
Recognition of Terminal 19-Base Pair Sequences ....................
pl Is a Cis-Active Transposase (and a Trans-Active Transposition Inhibitor) ....
Transposition MayBe Linked to Chromosome
Replication and~or Cell Division . .
Conclusior,~
............................................

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949
952
954
957
959
960

ABSTRACT

The bacterial transposon Tn5 encodes two proteins, the transposase and a
related protein, the transposition inhibitor, whoserelative abundancedetermines, in part, the frequency of Tn5 transposition. The synthesis of these
proteins is programmed
by a complexset of genetic regulatory elements. The
host DNA
~aethylation function, dam,inhibits transposase promoter recognition and indirectly enhancesthe transposition inhibitor promoter.The inhibitor
lacks the N-terminal55 aminoacids of the transposase, suggesting that this
sequence plays a key role in the transposition process. Anintact N-terminal
sequence is required for the transposase’s recognition of the 19-bp end DNA
sequences.This is the first critical step in the transposition process. Transposase-end DNAinteraction is itself regulated by an intricate series of
reactions involving several host proteins: DnaA,Dam,and Fis. The transposase is a uniqueprotein in that it acts primarily in cis and inhibits its own
activity in trans. Modelsto explain these properties are described. Finally
circumstantial evidencesuggests that transposition occurs preferentially from
newly replicated DNAthat has yet to be partitioned to progenycells. This
945
0066-4227/93/1001-0945502.00

Annual Reviews
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946

REZNIKOFF

timing of transposition is likely to havea selective advantagefor the host and
the transposable element.

Annu. Rev. Microbiol. 1993.47:945-964. Downloaded from arjournals.annualreviews.org
by University of Wisconsin - Madison on 10/19/05. For personal use only.

Introduction
Transposition is a recombination process in which DNAsequences termed
transposable elements movefrom an original site on a DNAmolecule to a
newsite on the sameor on a different DNA
molecule. In addition, transposable
elements can cause, and are associated with, other types of genetic rearrangements such as deletions, inversions, and chromosomefusions. The genomes
of prokaryotic and eukaryotic organisms contain these elements. One could
consider themas an ancient genetic machineryfor causing genomicrearrangements and, therefore, for facilitating genomeevolution. In addition, their
associated biochemicalreactions are likely to be similar to other interesting
events involving the interaction of proteins and DNA.For these reasons
transposable elements are of considerable interest.
The transposable elements found in Escherichiacoli fall into three general
classes determined by their mechanismsof transposition. Transposons such
as Tn3 and "~ transpose through a two-step replicative mechanismin which
a cointegrate (fused replicon) structure is an intermediate. TransposonsTnlO
and Tn903 transpose through a conservative cut-and-paste mechanism.
BacteriophageMuand related viruses represent the third class of transposable
element. In these cases the transposition can occur through either of the above
two mechanisms,depending upon the proteins involved and the precise nature
of the DNAstrand cutting after an intermediate is formed between the
transposable element and the target DNAsequence. Tn5 is generally assumed
to transpose via a conservative mechanism(2); however,this presumptionhas
not been critically tested. The reader is encouragedto examinethe models
and evidence for these transposition mechanismsin the recent monographby
Berg & Howe(3).
The conservative and replicative mechanismsof transposition share many
basic characteristics. The transposable element encodestwo critical functions
required for the process--the end sequences and a protein termed the
transposase. The element is defined by the specific sequences at its end.
Transposition and related events removethe transposable element from its
original sequence context precisely at the ends of these sequences. Changes
in any base pair of these sequences typically reduces the frequency of or
abolishes transposition. The transposable element also encodesa protein called
the transposase. Thetransposase is a critical participant in manytransposition
functions including: specifically binding to the end sequences, bringing the
twoends together through a protein oligomerization process, cutting or nicking
the DNAadjacent to the end sequences, and inserting the transposable element
DNAinto a DNAtarget site.

Transposition is. although some aspects are commonto all. However. The mechanismsof the regulation vary remarkably among the manycases studied.annualreviews. The transposase is a protein that performs multiple complexfunctions. Downloaded from arjournals. perhaps not surprisingly. Protein structure/function studies would seek answers to the questions about the organization of the peptide domains that perform these functions and how they interact. and transposable element end sequences maybe a perfect .org by University of Wisconsin . there are always surprising connections in biology.position process may give us insights as to how host DNA metabolismis organized and regulated. Workdone on other systems suggests that short DNA sequencescan dictate several extremely intricate reactions. are similar to ones found for other bacterial genetic systems. such compact complexity. The terminal DNAsequences are at first glance simply an exampleof a target for protein binding. Rev.. Understanding how they use these functions will tell us muchabout the transposition process and about the functionality of the host functions themselves. example of. Implicit in the above general description is the fact that several very interesting molecular events are involved in and regulate the transposition process. . For instance. Such tight regul~. transposable elementencoded-functionsoften play a critical role in this regulation. and the protein-end sequence interaction is associated with at least two events: protein binding and DNAstrand scission. Becausemanyof these host functions are ones involved in host DNA metabolism.their functionality is considerably more complexbecause they are often the target for more than one protein. Finally. elucidating howtransposition is regulated in a given systemwill tell us how complex DNAmetabolizing processes might be controlled and give yet more examples of how gene expression can be modulated. Studyingthese events will help us understandother genetic processes.In addition to the role of host functions.tion mightenable the host cell to strike a balance betweeninsuring proliferation of the transposable element and insuring the cell’s owngenetic survival--the very process of transposition causes chromosome breakage and rearrangements. if the host functions that Tn5uses are organized in the cell in a unique sp~ttial fashion.Annual Reviews www. in general. By studying transposition we embarkon a largely unknown path into the cell’s metabolism. TN5 TRANSPOSITION 947 Host proteins also play critical roles in the transposition process such as facilitating the end-sequence binding of the transposase. highly regulated process.org/aronline Annu. For personal use only. studying Tn5transposition mayreveal that arrangement. 1993. Transposable elements use host functions like biochemical parasites. and the strategies.Madison on 10/19/05.understanding their role in the tran.47:945-964. and regulating several steps in the transposition process.annualreviews. a quite rare. For instance. performingthe necessaryrepair or replication functions.. Microbiol. nucleating the higher-order structure in whichthe ends are brought together.

19. The relative abundanceof Tnp and Inh plays a major role in determining the Tn5transposition frequency. ISSORand IS50L(see Figure 1 for a schematic). Thus the regulation of these promoter activities becomesa crucial question. ISSOL andIS50R. while IS50L contains an ochre codonthat results in the synthesis of inactive proteins (33). The possible function of this sequenceis an importanttopic of investigation. Tn5is an exampleof a composite transposon in which antibiotic resistance genes are flanked by two nearly identical insertion sequences.IS50R encodes the transposase (Tnpor pl) anda second proteinthat inhibitstransposition (Inhor p2). BothIS50 elements are delineatedby19-bpsequences. TnpandInh are translatedin the samereadingframe. In cis it acts as a transposase. 19).Madison on 10/19/05.annualreviews. Inh’s only knownfunction is to inhibit Tn5 transposition. catalyzing the transposition of the Tn5 or IS50 sequences from which it was encoded (15. For personal use only. 1993. the insideend(IE)andthe outsideend(OE). Mylaboratory and several other investigators have been studying the bacterial transposable element Tn5as a model system. nonfunctional analogues of TnpandInh.in trans it primarily acts as an inhibitor of transposition (42). IS50Ris a fully functional transposable element. However.47:945-964. The Tnp inhibitory activity is one meansby which Tn5 transposition is down-regulated. 18. 18. separate. Downloaded from arjournals. and . 43). p4 IS50L IE kanr sttr bleo r IE ~ "~ ~. IS50Rencodes the transposase (pl or Tnp) (15. A secondprotein [the inhibitor (Inh or p2)] is also encodedby IS50R(15. As discussed below. and this review discusses our current understandingof this function. 19. Microbiol. promoters programTnp and Inh syntheses (21. which is thought to be the major meansof down-regulatingthis process. Interestingly. Rev. 45). apparently competing. 33).Annual Reviews www. Inh is translated in the samereading frame as Tnpbut lacks the N-terminal 55 amino acids. ’~ IS50R OE pl (Tnp) p2 (Inh) Figure1 Transposon Tn5.Tn5is a compositetransposonin whichgenesencodingthree antibioticresistanceproteinsare bracketed bytwoIS50elements.annualreviews. The paradoxof Tnpinhibiting the activity of other Tnpmoleculesis discussed in greater detail below.org/aronline 948 REZN1KOFF Tn5 OE p3 Annu.org by University of Wisconsin . IS50Lcontainsan ochrecodonthat results in the synthesisof p3 andp4.but the InhAUG is 55codonsdownstream fromthe TnpAUG. The properties of Inh relative to the Tnpsuggest that the N-terminal 55 amino acids of Tnp play an important role in its activity. and the Tnphas twoopposingactivities. it is the host that regulates the relative activity of these twopromoters.

Tn5 has also evolved a mechanismfor preventing the spurious synthesis of Tnpby virtue of accidental placementwithin an active transcription unit (21). Others will becomeobvious during the discussion of the lethal effect of overproducing Tnp. this review is ntot a comprehensivetreatment of the subject.annualreviews. whereasIS50 transposition uses an OEand an IE sequence. have been suggested above. The strategy accomplishthis translation control maybe widespreadand is discussed below. Rev. Tn5 sequences that were newlyintroducedinto a cell transposed at dramatically lower frequencies in a cell already containing Tn5as opposedto a cell lacking TnS. Howare the syntheses of Tnp and Inh regulated? 2. 1993. Twoof the relevant host functions (DnaAand Dam)also link the transposition process to DNA replication. Rather. Tnp binds to both of these sequences during the transposition process. Finally.Madison on 10/19/05. TN5 TRANSPOSITION 949 the regulatory mechanismthat has evolved appears to link the occurrence of transposition to the DNAreplication process. Whatis the molecularbasis for the cis-active nature of the transposase? 5. and they likely perform other functions vital for Tnpactivity. Therefore. The translation initiation signals were designedsuch that they will only function a:~ part of a correctly initiated TnpmRNA (21.In addition. Tn5 transposition utilizes two OEs. we are just nowbeginning to obtain clues as to the possible relationship of Tn5transposition with overall cell processes. In addition. Each IS50 is boundedby two unique 19-bp end sequences [termed outside (OE)and inside (IE) ends] that are critical for transposition (17. this chapter concentrates on areas of current and future research interest raised by the questions implied above. distinguisl~ting feature betweenIS50 and Tn5transposition is the choice of the transposable element ends. Microbiol.org by University of Wisconsin . they are the sequencesrecognized by host functions. Whatare the possible functions for the TnpN-terminal sequence? 3. The specific topics covered are: 1.Annual Reviews www. For personal use only.47:945-964. Howmight transposition be linked to chromosome replication and/or cell division? The Regulation of Transposase and Inhibitor Synthesis Studies by Biek & Roth (4) first indicated that the frequency of Tn5 transposition was regulated by Tn5-encodedfunctions. . Downloaded from arjournals. a host function binds to the OEto enhance transposition while the host functions affecting the IE down-regulatetransposition.annualreviews. which point to DNAreplication. 34).org/aronline Annu. Someof these clues. 37). Whatsort of protein recognition reactions occur at the 19-bp terminal sequences? 4. As we shall see. Douglas Berg (whose laboratory has contributed muchof what we know about Tn5) recently published an excellent review of Tn5(2).

Madison on 10/19/05.47:945-964. TheAUGs for TripandInh are indicated(21). Tnp and Inh are expressed from overlapping. Overlapping the TI .org by University of Wisconsin .to fivefold increase in T1 mRNA and a twofold decrease in T2 mRNA. and this change in transposition seems to mirror (and presumablyis the consequence of) a four. For personal use only. .~ (Inh) TI-35 GATCT(~ATC LexA? Figure2 Controlling elements for Tn5/ISS0 TnpandInhsynthesis. Between the -35and. see below) a consequence the Tn5-encodedInh protein.annualreviews. they also reveal genetic regulatory motifs found in other systems.the frequency of transposition is in part set by the abundanceof Tnp and the ratio of Tnp to Inh. Downloaded from arjournals.. Microbiol. and site-specific mutation studies prove.annualreviews. Rev. that the relevant GATCdarn T1 AUG (Tnp) T2 OE -. the T2 transcript can only encodethe Inh while the T1 transcript encodes both proteins [but the Inh is translated inefficiently from the T1 message(37)]. Thus the incoming Tn5 in the Biek & Roth experiment(4) encountereda preexisting pool of inhibiting Inh. Wenowknowthat this down-regulationis primarily (but not entirely. see 30) and can programcontradictory functions. 950 REZNIKOFF we (18) found that the Tn5 transposition frequency was constant regardless of the copy numberof Tn5(hence the transposition frequencyof an individual Tn5 decreased in the presence of additional Tn5s). 1993.10regionsof is a weakLexA bindingsite (23). As shownin Figure 2.The5’ endof IS50is defined bythe 19-bpOEsequence (see Figure4.Annual Reviews www.org/aronline Annu. The frequency of Tn5 transposition is 10-fold higher in damhosts.10regionare twoDam methylation sites that whenmethylated down regulateTnpmRNA synthesis(43). DNA(dam) methylation down-regulates the synthesis of the T1 (Tnp) transcript and appears to up-regulate the synthesis of the T2 transcript (43). but the amountof cis-acting Tnp per Tn5 remains constant. An inspection of the DNAsequence indicates. Suchoverlapping promoters are found in other systems (e. The opposite effect on promoter activity suggests that the promoters compete for RNApolymerase.~. Thus by studying this Tn5 arrangement we will elucidate a more general regulation strategy. Moreover. probably competitive. What then determines the abundanceof Tnp and Inh? The answers to this question are not only interesting for Tn5. as the copy numberof Tn5increases. TheT2(Inh) promoteroverlaps T1(21). Inh functions in trans to block transposition. promoters. . below).g. This conclusion was deduced by a deletion analysis of in vivo promoter activities (21). the concentration of trans-acting Inh increases.Thepromoter for transposase synthesis(T1) initiates transcription66bpfromthe endof IS50(21).

Production of these transcripts lmight result in spurious transposase synthesis. whichis exactly the observedresult (27.org/aronline Annu.annualreviews.:21. Other transposon systems [in particular TnlO (31)] also display Dam methylation control of Tnp synthesis and of transposition. thereby giving rise to read-through transcripts into the transposase gene. Tn5cou]td transpose into highly expressed genes. this regulation should stimulate transposition off of DNAthat is newly introduced into the cell. This mechanismreappears in the discussion of protein recognition of the IS50 IE sequence. 1993.47:945-964. Thestart of the T1transcript(which will notforma secondary structure)andthe TnpAUG are indicated.10 region of the T1 promoter. 32).org by University of Wisconsin . Microbiol.Annual Reviews www. For personal use only. Rev.these studies der~aonstrate in a quite precise mannera type of gene regulation displayed in many systems--chemical modification of specific DNAsequences to modulate protein binding.Thisfigureis similarto one shown in Ref.annualreviews.Madison on 10/19/05. GUC C-~ U _~u U IJCCCGUUUUCCAGG G U AACUUCUGCUCUU 110 40 Figure 3 Secondary structurein read-through transcriptsblocksTnptranslation. TN5 TRANSPOSITION 951 methylationsites overlap with the .a set of experiments(21) has demonstratedthat read-through expression of transposase does not occur because such messagesdo not programthe translation of Tnp. . However.The specific relevance of damregulation of Tnp(and Inh) synthesis is that it should couple transposition to DNAreplication (about which more is discussed below) because newly replicated DNAis hemimethylated.Transcripts that readthroughthe endof ISSOdonot expressTnpbecauseof a secondary structurethat occludes the Shine-Dalgarno sequence endandthe AUG (21. Also. 37). Downloaded from arjournals. Moreover.

Weinreich. Because Inh does not promote transposition. but not in the one which we studied. The former difference is a matter of technique. 45). For instance. M.annualreviews. binds to OE and IE end sequences specifically and shows the expected sensitivity to OEsequence mutations and Dammethylation of the IE (N. T. while the latter may suggest an interesting molecular phenomenon. The same mechanismappears to be shared by several other transposable elements [IS2 (13).org/aronline Annu. The Tnp. and (b) the sequence contexts of the Tn5 systems were different. suggesting that the N-terminal 55 aminoacids encodea domainof importance. Reznikoff. Jilk &W. de la Cruz. RNAsequences upstreamof the T1 transcript start site are necessaryfor the formationof this secondary structure. Recent studies have demonstrated that deletion of 11 . it is missingone or morecritical Tnpactivities (15-17. presumably by binding to a weak LexAbinding site upstream of the T1 promoter (23) (see Figure 2). 11). A. R.Annual Reviews www. while Kuan & Tessman postulate that LexArepresses transposase synthesis approximatelyfivefold. the samepattern is seen for lac (36. Reznikoff.org by University of Wisconsin . For personal use only. 952 REZNIKOFF An RNAsecondary structure in these read-through RNAsoccludes the Tnp translation initiation signals (37). Downloaded from arjournals. Wiegand. These studies differ in two respects: Weinreichet al performeddirect tests of a LexAeffect (40). This mechanismprevents read-through expression of Tn5 Tnp. IS4 (20). In vitro analyses of purified Tnp and purified Inh have shownthat at least one property missing in the Inh preparations is a DNAsequence-specific binding activity. 1993. 46) and gal (26)].47:945-964. Rev. and if so. The TransposaseN-Terminal Sequence Is Critical for Sequence-Specific DNABinding and Other Transposase Activities The transposase and its inhibitor are identical in sequenceexcept that the Inh is missing 55 N-terminal amino acids (20).Madison on 10/19/05.annualreviews. Do other mechanismsexist that regulate transposase synthesis? Recent reports present conflicting evidence regarding the possible role of LexAin regulating transposase synthesis. on the other hand.Many genetic regulatory proteins are enhanced in their DNA-bindingactivities through cooperative binding to a secondarysite (1). M. submitted. As shownin Figure 3. and TnlO (5)] and maybe a general motif for Escherichia coli gene systems [for instance. Krebs & W. IS3 (39). binding the lac repressor to its operator is thought to be stabilized through a bondto a secondary binding site. thereby forming a looped DNAstructure (10. 33. Microbiol. whethersuch a fortuitous nearby LexAbinding site could facilitate LexAbinding to its weakTn5 site. unpublished results). Our laboratory has found that LexAhas no significant control over transposase levels (40). but the Kuan Tessmanstudies were indirect (23). Future studies might examine whether such a nearby LexAbinding site is present in the Kuan-Tessmansystem. IS5 IS150 (38).

unpublished results). Downward-pointing arrowsdenotemutationsthat act like a deletion in the adjacent. Reznikoff.Annual Reviews www. 34. (Bottom)For the inside endsequence. Reznikoff.3’ (Fis) Figure4 (Top) Outsideandinside endsequences. TheTnp/Inhtelrminationcodonis the complement to positions 12-14. Damsite methylation regulatesbothTnpbinding(43) and binding(41). D. The11Achangegaverise to onedeletion in the secondclass.Madison on 10/19/05. Overproduction of the transposase (in the absence of transposition) kills host cells. Determining the precise DNA-bindingdomain will be instructive because the Tn5 transposase does not contain a previously classified DNA-binding motif..l¢f. These experiments show that the N-terminal 55 amino acids are necessary for the sequence-specific DNA-bindingactivity and imply that the DNA-binding domain is in this region.l l@ll | t~m~.org by University of Wisconsin .org/aronline Annu. 41). 1993. q?helower-caseletters indicate basepairs outsideof the required19-bpsequence. 44).annualreviews.47:945-964. Weinreich & W.TheDnaAbox is indicated for the outside endsequence (12. Microbiol. 17. Deletion of as little as three amino acids prevents this killing (M. A possible biological A i OE C 5’CTGACTC~~AGT (Dna A) 3’ C I E 5’CT GT CT C T T[~I’. Rev. For personal use only. Downloaded from arjournals. unpublished results). D. Upward-pointing arrowsdenotemutationsthat constitutethe secondclass in the adjacentdeletionexperiment(16). . and yet the activity is cltearly there.annualreviews.the Damsites andthe overlapping Fis bindingsite are indicated(34. Weinreich & W.-I |~-. TN5 TRANSPOSITION 953 N-terminal arnino acids or introduction of 4 different N-terminal missense mutations destroys the DNA-binding activity of Tnp (M. Another surprising property requires the N-terminal three amino acids of Tnp.deletion experiment.

1993. Jilk W. 34).Reznikoff. Figure 4 displays the distribution of bp defects. Jilk & W. T. All transposase-mediated genetic events (save one mentionedbelow) occur at the precise boundary the relevant 19-bp sequences. For personal use only. These are most easily detected in situations in which the OEfar removed(distal) from the location of the adjacent deletion defective. In addition. SomeOEpoint mutations (which totally block transposition) also fall into this class. The secondclass of adjacent deletion is unusual in that this type starts one bp removedfrom the OE. the protein-DNA interactions occurring at the OEand IE sequencesclearly display a remarkable degree of complexity. Twoclasses of adjacent deletions have been found. Tn5 is delineated by two inverted OEsequences and IS50 by an OEand an IE sequence (see Figure 4) (17. Krebs & W.If the distal OEis deleted. Downloaded from arjournals. However.annualreviews. These adjacent deletions are found in association with another subset of distal OEmutations. 29. A. submitted). Presumably the importanceof these sequenceslies in the fact that they are recognizedby proteins during the transposition process.47:945-964. N. Wiegand. greatly reducing or blocking transposition. and almost all changes in these sequences greatly reduce or abolish the frequencyof transposition (24. For instance.annualreviews. de la Cruz. Because . The OEend is recognized by both the transposase and the host protein DnaA(17.org/aronline 954 REZNIKOFF significance for the cell killing will be describedin the section on chromosome replication below. Tnprecognition of OEis fundamentalto the transposition process. different portions of the OEsequencemayplay roles in different steps in the transposition process. unpublishedresults). Microbiol.Annual Reviews www. somebase pairs could be recognized during the strand-cleavage reaction. Recognition of Terminal 19-Base Pair Sequences The Tn5 and IS50 transposable elements are defined by the terminal 19-bp sequences. Weinreich. Tnpcan catalyze deletions adjacent to its site of insertion starting at the end of the OEsequence. R. Rev. A.at least two proteins recognize the OEand three proteins recognize the IE. Mutant sequencesof this class that have been tested for transposase binding in vitro bind transposase with a lower affinity than the wild-type OE(R. 29). then the adjacent deletions start immediately adjacent to the functional OE. unpublishedresults). and the nature of the class dependsuponthe precise changein the distal OE. Reznikoff.org by University of Wisconsin . M. Tnp binds specifically to OEsequences in vitro as judged by gel retardation assays (42. 44). Annu. transposase recognition of the OE may be muchmore complex than a simple binding reaction. Althoughwe are not certain of all of the salient facts. someof which are only partially defective in transposition and do not seem to impair transposase binding in vitro (16. Reznikoff.M. The first evidence that various OEsequencesdictate different steps in the transposition process came from a complicated set of in vivo observations (16).Madison on 10/19/05.

It contains recognition sites for two pro~teins (transposase and DamDNA methylase). 44).sting that these base pairs maybe recognized by both proteins. showingthat Tn5and IS50 transposition occurs at a reduced efficiency in dna hosts. DnaA. Rev. DnaAal:~o recognizes the OEsequence. This Tnp-IE binding result is consistent with the observed transposition preferences (9. Jilk &W. The IE sequence is even more complicated. At positions 2 and 12 different mutations are associated ’with different classes of adjacent deletion formation--perhapsin these two cases the samebase pair is recognizedin different waysat different steps in tl~te transposition process. 14).Reznikoff. Fis.However. A similar complexityfor end sequenceshas beensuggested for other transposable elements (7.org by University of Wisconsin . and also encodesthe termination codonfor the transposase (see Figure 4).annualreviews. Later experiments tested the relevance of this binding.Thisfine structure is also seen in the binding of Tnp to the OE.org/aronline Annu. we could hypothesizethat the positions that differ are not recognizedby Tnpeither because they do not play a critical protein-rec- . and different base pair changes maydiscriminate differential]ty betweenthese two protein-recognition processes. 1993. sugge. Alternatively. Microbiol. This observation was first made through footprinting studies of the OE(12). 11A) are within the DnaA box. For position 2. yet the transposase binds in vitro to an unmethylatedIE with an affinity resemblingits binding to the OE(R. some mutations that presumably alter transposase binding (SG.this explanation does not fit somecases (2A. A closer examination of the results summarizedin Figure 4 suggests an additional level of complexity. For personal use only. unpublished results).47:945-964. On the other hand. A. flais implies a functional fine structure to the OE. Tnp might make different molecular contacts with the same base pair at different steps in the overall reaction. SomeOEmutations disrupt Tnp binding while others do not. The IE sequence and the OEsequence differ at out of 19 positions. and that a Tn5 derivative with a mutation in the OEDnaAbox did not appear to manifest sensitivity to the host dna genotype (29.Annual Reviews www. 25). Downloaded from arjournals. someend-sequence base pairs are recognized in very complexways. it overlaps a binding site for a third protein. 44). 3C) because the mutations are located outside of the DnaAbox as defined by sequence homology (12. This result is quite surprising because it :~uggests considerable flexibility in the DNA recognition domain of Tnp. Thus.annualreviews. TN5 TRANSPOSITION 955 different subsets of OEpoint mutationsgive rise to different classes of adjacent deletions. A possible explanation for someof the mutations that do not i~mpairtransposase bindingis that they are recognizedby a different protein. 10T. at different steps in the transposition reaction and/or by different proteins. Possibly both DnaAand transposase recognize position 12.Madison on 10/19/05. Current experiments are examiningthe effect of DnaA transposase binding and are attempting to determinethe in vitro properties of DnaAbindling to various OEmutant sequences.

The 24-bp IE construct demonstrates muchlower transposition frequencies in a damhost (9. indicating a direct effect of Dammethylation on IE recognition during transposition (24. and that Fis acts to inhibit transposition in a Dam-dependentmanner(41). 29.or a 24-bp sequence was used for IE. A. Jilk &W. it mayact to dampenIS50 transposition from newly replicated DNAduring the exponential phase. Werecently showed that Dammethylation of IE DNAfragments reduces transposase binding in vitro (R. transcription initiation programmedby the T1 (transposase) promoter directly inhibited by DamDNAmethylation of sites overlapping the -10 region (43).annualreviews. whenit is most abundant. DNAmethylation regulation of transposition maysuggest a coupling between transposition and DNAreplication.Madison on 10/19/05. Transposition of IS50 is sensitive to DamDNAmethylation because of the inhibition of two separate sequence-specific protein-DNAinteractions.org by University of Wisconsin . As described above. but because Fis abundancevaries with the stage of bacterial growth. 956 REZNIKOFF ognition role [position 4 maybe an exampleof this (24)] or because they are recognized by someother protein (e. 25).g. This observation is consistent with direct DamDNAmethylation control over transposition reactions using the IE. DamDNAmethylation inhibits transposase binding to the IE. As noted below. The role of DamDNAmethylation regulation of IS50 transposition (utilizing the IE) is complicatedby the fact that a secondprotein (Fis) binds to a sequence overlapping the IE. Microbiol. These studies gave quite different results depending upon whether a 19. DnaA).47:945-964. For personal use only. Downloaded from arjournals. unpublished experiments).the in vivo adjacent deletion studies suggest that mutations at someof these dissimilar positions do reduce Tnp binding directly (16).DamDNA methylation also inhibits IS50 (but not Tn5) transposition even when the transposase synthesis is programmedby a Dam-insensitivepromoter (tac).org/aronline Annu. However.However. Wenowknowthat the 24-bp sequence (but not the 19-bp sequence)contains the overlappingFis-bindingsite. Rev.Annual Reviews www.Reznikoff. 43).annualreviews. 1993. Thus we conclude that Tnp specifically recognizes two quite diverse sequences--another reflection of the complex substructures of IE and OE. This effect is presumably mediated through the Damsites within the IE (see Figure 4). In vitro gel retardation and footprinting studies confirm that Fis binds to this sequenceand that it binds efficiently only to the unmethylatedsite (41). The physiological significance of this Fis effect is unclear. Verysimilar effects of DNA methylation that regulate TnlO/ISIOtransposition have also been observed (31). . Somein vivo experiments designed to study the frequency of IS50 transposition actually used various recombinant DNAconstructs that have the same general structure as IS50 (OE-transposase gene-reporter gene-IE) but differ in the size of the IE sequence. and this binding (which is also blocked by DamDNAmethylation) is thought to compete with transposase-IE binding.

org/aronline TN5 TRANSPOSITION 957 Thetranslation termination codonfor the transposase (and its inhibitor) located within the IE at positions 14-12. For personal use only. by and large. The Tn5 transposase functions pdmarly in cis (14. The Tn903 transposase is knownto be chemically unstable (8). a low-abundanceactive conformation similar to the tethered protein and a high-abundanceinactive conformation. acting preferentially on OE-OE or OE-IEsequences located close to the site of transposase synthesis on the samerelicon (15. Downloaded from arjournals. The released completed Tnp is proposed to have two conformations in equilibrium. andTnp-Tnp or Tnp-lnh oligomerization prior to end-sequence bindingwill alsoinactivateTnp. but various studies have indicated that Tn5Tnpis. Wepropose that the nearly complete. 18). This observation has also been madefor the transposase proteins enc.oded by other transposons such as TnlO and Tn903(8. Once Tnpsynthesisis complete.org by University of Wisconsin . This preferential cis activity could result from either a chemical or functional instability in the transposase.annualreviews. Wehaveno information as to whether this influences transposition utilizing an IE or whetherTnpboundto the IE can influence the synthesis of the transposase. Presumably. 1993. Annu. Rev. 19).47:945-964. and/or froma sequestration of the transposase during or shortly after synthesis. chemicallystable in vivo (see 33). by the release of the Tnp protein from the translation apparatus and by the oligomeriz~:tion of Tnp with monomersof Inh or Tnp. The Tnp-Trip Oligomer ~ completion of translation incomplete tethered transposase free transposase ACTIVE ACTIVE free transposase [~~ INACTIVE Tnp-Inh Oligomer INACTIVE Figure 5 Transposase acts in cis. the molecule favorsan inactiveconformation. Sucha propertywouldobviouslylead to cis but not trans activity. Furthermore. tethered Tnp has a high affinity end-sequence binding activity and can initiate the transposition process prior to completionof its translation. Mycurrent hypothesis(pictured in Figure5) is that the Tn5Tnpis cis-active owingto protein conformationchanges that are regulated by two factors. Tnp tethered to the translation apparatus can initiate the transposition reaction.annualreviews.Madison on 10/19/05. . Microbiol. the formation of Tnp-Tnpor Tnp-Inh oligomers probably occurs with the inactive conformation(or oligomerization inactivates the active conformation).Annual Reviews www. 28). pl Is a CJis-Active Transposase (and a Trans-Active Transposition Inhibitor) TheTn5transposase functions primarily in cis.

For personal use only. 958 REZNIKOFF longer a Tnp monomerexists without end-sequence binding the higher is the probability of oligomerization. Also suggesting that Tnp activity is regulated by oligomerization is the observation that Tnpitself inhibits transposition in trans. The proposed model includes aspects of sequestration (the incomplete tethered Tnpis held in close proximity to its gene) and functional instability (a conformational change facilitated by oligomerization). M. Alternatively. de la Cruz. M. M. Downloaded from arjournals. Twosimple modelsexplain this inhibitory activity. Krebs & W.47:945-964. 19. submitted). Microbiol.Reznikoff. Inh might bind to the end sequences forming inactive Inh-end sequence complexes. Theprimary evidence supporting the notion that the tethered nascent protein maybind end sequences with an enhancedaffinity camefrom gel retardation experiments examining the binding of Tnp subfragments to OE DNA. This observation is consistent with the OE-bindingdomainresiding at the N terminus (see section 2 of this review) and suggests that this OEbinding domainis occluded in most of the intact Tnp. submitted). These mutant proteins may have altered the equilibrium between active and inactive conformationsof Tnp. Rev. Reznikoff.Madison on 10/19/05. T. 45).An in vitro synthesized Tnp subfragmentlacking 100 carboxyl terminal aminoacids binds specifically to OEDNAand does so with an affinity -10-fold higher than that of full-length Tnp(L.Inh and other N-terminal deletions of Tnp cannot bind end-sequence DNAefficiently (N. Wiegand&W. Wiegand. Mahnke&W. de la Cruz. 18.org by University of Wisconsin . but several of its predictions are clear. unpublished results). The Inh protein is knownto inhibit transposase activity in trans (15. Reznikoff. This mutantis fully functional for transposition in cis but inhibits transposition in trans. Reznikoff. and the mutations are believed to have a conformational effect because the mutant Tnp proteins are more sensitive to proteolytic degradation (T. The evidence for this modelis quite circumstantial. However. However. thereby blocking Tnp access to these sequences. The MA56 mutant destroys the Inh start codon and introduces an alanine in place of methionine at codon 56 of Tnp.org/aronline Annu. Thus this property wouldalso lead to cis activity. M. T. Weinreich. Wiegand. unpublishedresults). This property of Tnpwas discovered during an in vivo analysis of a Tnp mutant that fails to makethe Inh protein (42). Althoughthe modeldescribed above and in Figure 5 is consistent with all . 1993.annualreviews. In vivo studies suggest that protein oligomerizationregulates Tnpactivity. Wehave recently described Tnp mutants that increase the frequency of transposition both in cis and in trans (42). thus this modelis not correct. Evidencesuggests that Inh does oligomerize with Tnp and that the oligomers do have altered DNA-binding activity (N. Inh could form mixedoligomers with Tnp and alter Tnp activity. Weinreich.Annual Reviews www.annualreviews. Krebs &W.the completed Tnp is not completely inactive as Tnp does have some trans activity.

DeLong& Syvanen (6) reported that a small fraction transposase is associated with the inner membrane fraction upon cell lysis.org by University of Wisconsin . 44). unpublished results). unpublishedresults). manyof the host functions participating in Tn5transposition are assumedto be componentsof a sequestered organelle involved in host-chromosome replication. Weinreich & W. transposition does occur in the mutants resistant to transposase overproduction (M. The one that has been most closely examinedmapsnear thefts locus at 76 min (M. Downloaded from arjournals. Rev.47:945-964. do the hypertransposing mutants alter the conformation of Tnp? Will tethered. Cell death occurs in the absence of transposition as well as any other Tn5-encodedfunction (such as Inh or the end sequences) (M. Reznikoff. SulA:a protein that inhibits cell division.org/aronline TN5 TRANSPOSITION 959 the observations. Weinreich &W. D. 1993.Madison on 10/19/05. For instance. Howis ~this coupling accomplished and what is the advantage of this arrangement? Wehope an analysis of the mutants mentioned above will suggest an answer to the first question. D.at least the link to chromosome partition can be bypassed. Microbiol. indicating a failure in partition-dependentseptation. In particular the following host functions are knownto influence the frequency of Tn5transposition: Dam:a protein catalyzing postreplicative DNAmethylation.annualreviews. incomplete Tnp bind to OEDNAwith a high affinity? Does oligomerization favor the inactive Tnp conformation? Annu. dnaAcells have reduced transposition frequencies.annualreviews. Transposition May Be Linked to Chromosome Replication Somecircumstantial evidence might link Tn5 transposition to the process of chromosomereplication and partition. E. direct experimentsare neededto test it. col~i mutants resistant to transposase overproductionkilling continue to divide whentransposase is overproduced. D. Onepossi- . unpublishedresults). For personal use only. These results point to a possible link betweentransposition and chromosome-partitionfunction. fts genes encodefunctions required for septation. DnaA:an oriC-binding protein required for the initiation of DNAreplication (29. Weinreich. In addition. This link is in additionto the preferential transposition of Tn5 or IS50 sequences off of newly replicated DNA(which results from damregulatory effects) and to the sharing of DnaAin both transposition and replication processes.Annual Reviews www. Thesecorrelations are intriguing but do not showa mechanistic link. damcells have elevated transposition frequencies (43). However. As for the second. The dying cells form long multinuclear filaments. Reznikoff. sulA cells haveelevatedtransposition frequencies (35). Observations that suggest a functional link between transposition and chromosomepartition camefrom experiments analyzing the cellular consequences of transposase over expression. The mutations mappedusing Hfr crosses reside at more than one locus.

we find a complex Tnp-specific sequence. The work to date has elucidated (and future work will continue to elucidate) the mechanismsby which transposition occurs and is regulated and will also continue to provide insights into important aspects of molecular biology. an imperfect symmetryelement that blocks the expression of read-through mRNA. a possible LexAbinding site. the Tnp presumably executes the following operations: binding of OEand IE.org/aronline Annu. For personal use only. If.g.annualreviews. This element encodes all of the Tn5 functions required for transposition and all of the Tn5 regulatory mechanisms. Rev. and inserting the . 1993. translation initiation signals for Tnp and Inh.Madison on 10/19/05. a DnaA-bindingsite. e. assuring that the ongoing chromosome replication wouldbe completedprior to cell division.org by University of Wisconsin . I havenot discussed the bulk of the protein-coding sequencesin this review because the required Tnp(and Inh) structure-function studies have not been performed.Annual Reviews www. cells that contain transposase. although cis-active sites mayalso be in this region [e. Thustransposition would be biased towards cells in which a copy of the parental genomeis maintained and can be inherited. cleaving target DNAto give 9-bp 5’ overhangs. a promoterfor Inh mRNA synthesis. For instance. One of the interesting aspects of Tn5molecular biology is the density of information encoded within a short sequence. the proposed coupling of Tn5 transposition to chromosome replication and partition might decrease the risk of genomic "suicide" occurring as a result of the conservative cut-and-paste transposition process.annualreviews.Thus in the first 259 bp. Dammethylation sites regulating the Tnp promoter activity.a promoter for Tnp mRNAsynthesis. Microbiol. Most of the rest of the sequenceup to the Fis site. Conclusion The Tn5/IS50 system has been an amazingly productive object of experimental inquiry. Downloaded from arjournals. transposase inhibits chromosomepartitioning. Transposition is most likely to happensoon after DNA replication (whenthere are two copies of the donor DNAsequence) due to the influence of damDNA methylation on transposase synthesis and inside-end usage. bringing the ends together (or inactivating the unbound Tnp) through oligomerization. a Fis binding site of unknown function (41)]. 960 REZNIKOFF ble advantageto Tn5accruing from such an association is that a relationship between Tn5 and the same organelle might have evolved in order to insure that the organelle’s transposition apparatus wouldhave efficient access to these functions.g. The IS50 sequence is 1533 bp in length. whichoverlapsthe IE. Alternatively. is involved with encodingTnpand Inh. cutting the DNAnext to the end sequences. however.47:945-964. starting at the OE. then this might delay cell division in the very cells in whichtransposition is mostlikely to haveoccurred. and sequences encoding the important N terminus of Tnp.

. 1989. much work is left to be done. The author is indebted to the many members of his laboratory who have . W.. Moldave. Microbiol. how does DnaA binding to the OE influence the Tnp-OE interaction? How do the different base pairs of the OEinteract with Tnp and at what steps of the transposition reaction does this occur? Howdo the different base pairs of the IE function with regard to Tnp activity? Howdoes Fis perturb the Tnp-IE interaction? What is t~. Bacteriol. N. Syvanen. Berg.K. and how many molecules are involved in synaptic complex formation? Finally. Berg. Berg. M.E.. B. N. Mol. These and other questions will be the object of future experiments. Davi~. M.Regulation of Tn5 transposition in Salmonella typhhnurium. proteins of IS50R. In Progressin Nucleic Acid i~esearchandMolecularBiology. Role of instability in the cis action of the insertion sequenceIS903transposase. Where are they? Howare they organized in a three-dimensional structure? Understanding the molecular basis of Tnp cis activity should also reveal fascinating insights into this protein’s structure-function relationships. Muller-Hill.Washington. 9. Sci. 10.annualreviews.org/aronline Annu.x~ng.R. E. D. 185-210 3. L. von Wilcken-Bergmann. the NSF (DMB-9020517)... Howe.B. R. Soe. 1990. and the editor of this volume for very helpful suggestions.Annual Reviews www. Hwang. Record.Sci. D. USA77:6047-51 5. 3. ed. D. USA87:4048-52 Dodson.Madison on 10/19/05. Grindley. and structural analyses. 1989. Genetic analysis of the interaction of the insertion sequenceIS903transposasewith its terminal repeats. 172: 5516-19 Derbyshire. D. Simons. Membrane association of the Tnpand Inh 7. J.Each of the functions requires particular Tnp domains of critical importance. M. K.annualreviews. Proc. which will require detailed genetic...47:945-964. Even among the transposition operations for which we have substantial information.Proc.39:81-128. M.Cell 43:379-97 6. 195:949-52 . 1985. 1990. R. M. Kleckner. TransposonTn5. TN5 TRANSPOSITION 961 transposab]~e element into target DNA. Mobi. Acad. Acad.. F. DNAloop formation: role in generegulation and implications for DNA structure. Rev. K.A. Natl. For instance. M. DC: Am. F.contributed their imagination and work. See ~tef. Acad. Sci. Biek.!e DNA. Roth. D. T. W.. 1987.Specific destruction of the secondlac operator decreasesrepressionof the lac operon in Escherichiacoli fivefold. K.. Downloaded from arjournals. Microbiol. research efforts should be directed at determining if transposition is coupled to the cell-division cycle and how that linkage is accomplished. D.J. G. inverted USA84:8049-53 Derbyshire. N. and the American Cancer Society (MV-554). Biol. Literature Cited 1.. 1993. M. biochemical.J. E.M. Kramer. For personal use only. Natl... 1987.Tnl0protects itself at two levels fromfortuitous activation by e~:ternal promoters.. 8. Factorsaffectingtranspositionactivity of IS50 and Tn5ends.te active form of the Tnp. Jr. 1989. NewYork: Academic 2.A. Gene76:207-13 Eismann. pp. Del. Grindley. W.Cohn. 1990. 1990. Bellomy. E.Proc. 4. ACKNOWLEDGMENTS The research in the author’s laboratory has been supported by grants from the NIH (GM19670).org by University of Wisconsin .Natl.

Mol.. S. Hoopes. E. 1993. 147: 12. 1991. Rev. 34. C. Aead. 1988. W. M. 84:9118-22 M... Reznikoff. W. Fuller. LexA mRNAfrom lacZ translation initiation protein of Escherichia coli represses mutants. Cell 43:117-30 1984. StrucRes. Rezrfikoff. Ghosal. J. P. 38. L. Bacteriol. P. W. C. J. Biol. 33. C. 1985. Proc. Regulation of Tn5 by the 30. Downloaded from arjournals. N. Genet. Syvanen. S. L. Commun.. Biophys. tion... 211:427-45 expression of the Tn5transposase gene.47:945-964. Mol. 1987. I. M.. Berg. Biol. H. C. Reznikoff. Tu. USA 80:7293-97 21. Mol. J. Huisman. H. Roberts.. Burgess. W. Syvanen.. L... The role of the ISSORproteins 32. W. The functional differences in the of Tn5transposition in Escherichia coli inverted repeats of the Tn5are caused is mediated by protein from the right by a single base pair nonhomology. For personal use only. S.-T. W. S. 1983.. 1983. J. R. Kroger. C. Y.. Bertrand. Reznikoff. tive origin (oriC) and other DNA sites. Nature 304:280-82 ISI0 transposition is regulated by DNA 18.-J. Makris. nucleotide 25. B. Sasakawa. 14. pp. Merril. Starlinger. 1983. transposition.. C. J. D. Krebs. 1981. 171: Nucleic Acids Res. P... Nucleotide sequenceof the transacts preferentially on nearby transposon posable DNA-element IS2. J. F. J. Y. H. R. G. S. M. E. C. W. Isberg. S.. P.. Altman. 173:6406-10 Translation initiation of ISSORread24. Natl. Kinetics of Tn5 transposition... Nature 297:159-62 In vitro secondarystructure analysis of 23. 1990.. Reznikoff. Bacteriol. Acad. Komberg.. Reznikoff. S..G. Mctransposon Tn5required for transposiClure. J. The dnaA protein complex 27. Mutational 65-80 analysis of IS50 and Tn5 ends. R. Johnson. S.. Yin. P. R. Johnson. Tillmann. coli insertion element. 1982. Wickens. R. M. Acad. 16:6789-99 5212-14 39.. 1989. Krebs. 1991. The position at the termini of ISS0. W. C. Dahiberg. S. Gralla. 170:889-94 Cell 38:889-900 28. D. Microbiol. A. K.. Kroger. Way. O. Maquat. and transposition. 1984... R. Record. C. Jilk. Gen.. 1982. C. S. V. Carle. R. S. 35. P. Schnlz. B.. Rak. 1984. 37. S. L. M. Natl. M. Klaer. H. 1989. Young. N. Johnson. repeat. J. Proc. Kuhn. 192: of Tn5 from ~. J. R. E. with the E. W. D.-P. Morisato.. Berg. 1993. Reznikoff. Proc. in the promotion and control of Tn5 R. Sci. adenine methylation. 1979. Proc. Lazaar. K. J. 875-87 A.annualreviews. O. D. D. W. Flashner.. R.org by University of Wisconsin . Funnell. Implications of Tn5 associated adjacent Gross. Saedler. Mol. C. W. C. V.. Reznikoff. 781-91 22. Sommer.. J. Tnl0 transposase 13. DNAsequences at the ends of 31. 962 REZNIKOFF 11. S. E. 1981. P. Hobom.. Kuan. W.. J...S. 1987. Acad. Cell 30:873-82 Cell 23:191-99 20. et al. Natl. Escherichia coli in the lac operon.. sequence of IS4.. Control of transposase and affects the efficiency of transposition inhibitor expression. A. R. A. M. Signon. Kim. W. lon-sulA regulatory function of IS50. of Transcription. E.-P. C.org/aronline Annu. R. . 8:2101-9 sition. Makfis. M.. McCommas. Yoshikawa. Phadnis. 1982. Jr. IS150: distribution. E.annualreviews.-J.Annual Reviews www. 105-13.. 177:64. USA 15. Kleekner.. Errada. Cell 32:799-807 Acids Res. 6:1111-22 29. S. Sequencesessential for transFritz. Sasakawa. 1986. The control 1981. Dual 26. T. E. Reznikoff. L. 175:1264-71 Elsevier 17. 142:879-84 tural analysis of insertion sequenceIS5. Bacteriol..J. 36. In RNAP olymerase and the Regulation Cell 30:883-92 16. Mol. A. Gottesman. C. Rothstein. R. b221 ci857 Pam Oam to the chromosome. R. Nordmann. mechanismof repression at a distance Adhya. Rosetti.. 181:169-75 Natl. Timmerman. level of the transposition reactions? E. N. Transcriptional and translational sites M. E. Schwartz.Madison on 10/19/05. right repeat proteins: control at the Donnelly. New York: deletions. P. 1987. Reznikoff. Sci. Schniz. Kleckner. Nucleic ends in vivo. Identification of base pairs in the outside end of insertion sequence ISSO that L. Makris.. L. J. B. EMBO are needed for IS50 and Tn5 transpod. Reznikoff. USA 85:8968-72 lambda induction. sequence and phylogenetic relationships Orientation of IS50 Iransposase gene of a new E.Biochera. Uno.. C. through transcripts. eoli chromosomalreplieaTemporalcontrol of Tn5 transposition. USA 85:2224-28 1988. ed.. R. C... Mutational analysis of ISl0’s outside end. S.5-61 Gene 32:91-8 19. Kleckner. Biol. Biol. Sci.I. D. S.. D. Tessman. 198. 221: Reznikoff.. ..H. Complexpromoters. S. ~’. 1988.. Bacteriol. Borchardt. Bacteriol. gal operon proteins madeafter prophage Sci..

an essential host gene. Characterization of two hypertranslx~sing Tn5mutants. W. C. TN5 TRANSPOSITION 1985.. S. S. X. 199:35--46 44. W. Weinn. Biol.. p2 and the inhibition of Tn5transposition. 40.. Wieg~md. S. Molecular cloning and sequenceanalysis of trp-lac fusion deletions.’ich. W. Downloaded from arjournals. Reznikoff. 169:4637-45 45. J. 1987. Bacteriol. S. Mol. J.. J. Bacteriol. W. 172:355-62 . and Tn5 mutants. Induction of the SOSresponse in Escherichia coli inhibits Tn5and IS50 transposition. J. S. W. M. 1993. P. C. 1992. J. J.-P. 174:453037 42. 1988. Reznikoff. Reznikoff.. W. Reznikoff.Madison on 10/19/05.. M. 173:6910-18 41. T. Microbiol.Annual Reviews www. Reznikoff. Fis plays a role in Tn5 and IS50 transposition.-P. C.annualreviews.-P. Bacteriol. dnaA. J. Biol. Munson. Rev. Mol. Krebs. S. S.annualreviews.. 1991. 13:2127-39 Weinn.org by University of Wisconsin . J.47:945-964. M... Yin. 1988. W. Makris. W. 170:3008-15 46. M. Complete sequence of IS3. Nucleic Acids Res. Yin. For personal use only. The effect of dam methylation on Tn5 transposition. Yu. D. Yin.qch. C. J. Reznikoff. 1992. Bacteriol. J.L. J. M. 174:1229-39 963 43. Reznikoff.org/aronline Annu. D.. 1984. Bacteriol.

Annu. For personal use only. . Downloaded from arjournals. Rev. Microbiol.annualreviews.47:945-964.Madison on 10/19/05. 1993.org by University of Wisconsin .

Microbiol.Madison on 10/19/05. Rev.annualreviews.org by University of Wisconsin . For personal use only.47:945-964.Annu. Downloaded from arjournals. 1993. .