Laboratory of Molecular and Cellular Biology, National Institute of Diabetes Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
Correspondence
Deborah M. Hinton
dhinton{at}helix.nih.gov
![]() |
ABSTRACT |
---|
![]() ![]() ![]() |
---|
Present address: Center for Advanced Research in Biotechnology, University of Maryland, Rockville, MD, USA.
Present address: Trinity University, Washington, DC, USA.
||Present address: Institute of Systems Biology, Seattle, WA, USA.
¶Present address: Uniformed Services University of the Health Sciences, Bethesda, MD, USA.
Overview
Upon infection of Escherichia coli, bacteriophage T4 establishes its own developmental cycle. Within 20 min, the phage programmes the generation of approximately 200 copies of its genome, the packaging of that DNA, and finally its escape from the host by lysis (reviewed by Miller et al., 2003). Regulation of this cycle is achieved largely by phage promoters, which sequentially express early, middle and late phage genes. Because T4 does not encode its own RNA polymerase, it must direct the host transcriptional machinery to these phage promoters at the correct time during infection. T4 encodes factors that accomplish this takeover by altering the specificity of the host E. coli RNA polymerase as infection proceeds (reviewed by Miller et al., 2003
; Stitt & Hinton, 1994
).
E. coli RNA polymerase consists of a core of five subunits (2,
,
' and
), which contains the RNA-synthesizing activity, and a
factor that binds to a specific promoter sequence and sets the start site for transcription (reviewed by Gruber & Gross, 2003
; Paget & Helmann, 2003
). The primary
,
70, is the predominant recognition factor for the transcription of housekeeping genes. Other
factors are used under certain growth conditions or at times of stress. RNA polymerase containing
70 is the major polymerase species present during exponential growth, the condition under which T4 infects. When present in polymerase,
70 usually recognizes two regions in promoter DNA: a 10 element, having a consensus sequence of 5'-TATAAT-3', and a 35 element, having a consensus sequence of 5'-TTGACA-3' (Campbell et al., 2002
; Gardella et al., 1989
; Keener & Nomura, 1993
; Murakami et al., 2002b
; Siegele et al., 1989
; Vassylyev et al., 2002
; Waldburger et al., 1990
). (All sequences are given as the top, i.e. the non-template, strand of DNA.) To a first approximation, the strength of a host promoter reflects the match between its 10 and 35 sequences and the canonical sequences for these regions. A subset of
70-dependent promoters lack a good match to the canonical 35 consensus sequence, but nevertheless are recognized well by polymerase. These promoters, designated extended 10 promoters, contain the sequence 5'-TG-3' at positions 15/14 and thus have an extended 10 sequence of 5'-TGnTATAAT-3' (Bown et al., 1997
). In addition, contact between the
subunits of polymerase and the sequence between positions 40 and 60 (UP elements) can contribute to promoter recognition and strength (Ross et al., 2001
; Ross & Gourse, 2005
).
At the start of infection, early T4 promoters must compete with host promoters for the available RNA polymerase. T4 wins this competition with two main strategies. First, early promoters contain excellent matches to the canonical 10 binding element and although their 35 sequences differ somewhat from the host canonical 35 element, these promoters are still recognized well by host polymerase. In fact, when present on a plasmid, these T4 early promoters are stronger than excellent host promoters (Wilkens & Ruger, 1996). In addition, T4 injection of the phage Alt protein (reviewed by Miller et al., 2003
; Stitt & Hinton, 1994
), which ADP-ribosylates one of the
subunits of the core, immediately increases recognition of the phage early promoters over host promoter sequences (Sommer et al., 2000
; Wilkens et al., 1997
). Thus, the strength of the T4 early promoters together with the modification of the
subunit ensures that the transcriptional machinery of the host switches very quickly from host DNA to the T4 genome.
Phage middle promoters become active about 1 min after infection at 37 °C (reviewed by Brody et al., 1995; Stitt & Hinton, 1994
). These promoters contain the
70 10 DNA element and are dependent on RNA polymerase containing
70 for transcription, but they lack the
70 35 DNA element. Instead they have a different consensus sequence (a MotA box, 5'-atTGCTTtA-3') centred at 30 (Marshall et al., 1999
; Stitt & Hinton, 1994
; Truncaite et al., 2003
). Activation of middle promoters requires a T4 transcription activator, MotA (Hinton, 1991
; Mattson et al., 1974
), and a T4 transcription co-activator, AsiA (Hinton et al., 1996b
; Ouhammouch et al., 1994
, 1995
; Stevens, 1973
). By itself, AsiA also functions as an inhibitor of
70-dependent transcription (Stevens, 1972
, 1973
). In addition, during middle gene expression, a second ADP-ribosylating enzyme, the T4 Mod protein (Tiemann et al., 2004
; reviewed by Miller et al., 2003
; Stitt & Hinton, 1994
), ADP-ribosylates the other
subunit of RNA polymerase. Thus, by about 4 min after infection, both
subunits are fully modified. ADP-ribosylation may serve to decrease the strength of host promoters (Miller et al., 2003
) by eliminating the ability of the
subunit of polymerase to bind to promoter UP elements present in the 40 to 60 region of some host promoter sequences (reviewed by Ross et al., 2001
). In addition, by this time of infection, multiple phage-encoded nucleases are digesting the host DNA, eliminating the supply of any competing host promoter sequences and further ensuring the commitment to phage transcription (reviewed by Carlson et al., 1994
; Miller et al., 2003
). Phage DNA, which contains glucosylated, hydroxymethylated cytosines, is immune to this nuclease action.
T4 late transcription (reviewed by Miller et al., 2003) still requires host RNA polymerase core, but the
70 subunit is replaced by a
factor composed of the T4 gene products 55 and 33 (Kolesky et al., 2002
; Nechaev et al., 2004
). Polymerase containing this alternative
, in the presence of the late transcription activator T4 gene product 45, recognizes and initiates transcription from phage late promoter sequences. 45 protein is also the DNA polymerase clamp (Moarefi et al., 2000
; Nossal, 1992
), and the activation of late promoters requires active DNA replication. This connection of late transcription to the replication of phage DNA serves to link the expression of late genes, whose functions are primarily DNA packaging and capsid assembly, with the level of phage DNA.
In this review, we focus on the activation of transcription from T4 middle promoters. As detailed below, the switch by polymerase from recognizing host and T4 early promoters to T4 middle promoters involves retaining some of the 70DNA contacts found in host promoter recognition while rearranging others. This type of activation is called
appropriation.
The 70 subunit of E. coli RNA polymerase and recognition of host promoters
70 belongs to a large group of bacterial
factors (Gruber & Gross, 2003
; Lonetto et al., 1992
). Like all primary
proteins, it can be divided into four main domains (regions 14; Fig. 1
, top), which are further subdivided into subregions (1.1, 1.2, etc.). Two of these subregions, region 2.4 located in the middle of
70 and region 4.2 located in the C-terminal portion of the protein, recognize host promoter DNA (Fig. 1
). Crystal structures obtained using promoter DNA in a complex with Thermus aquaticus RNA polymerase (Murakami et al., 2002a
) or a 35 DNA element in a complex with region 4 of the primary
of T. aquaticus (Campbell et al., 2002
) have demonstrated that residues in an
-helix in region 2.4 are responsible for contacts with the 10 DNA element while region 4.2 contacts the 35 promoter element through a classic helixturnhelix motif (H3-T-H4 in Fig. 2
) Residues within region 4 that contact the 35 element DNA in the crystal structure of region 4 with this DNA are indicated in magenta in Fig. 2
. Amino acid substitutions at
70 residues R584 (Gregory et al., 2005
; Siegele et al., 1989
), E585 (Keener & Nomura, 1993
) or R588 (Gardella et al., 1989
) render
70 better able to use promoters with specific base-pair changes at positions 31, 33 or 33, respectively, suggesting that these residues directly interact with base moieties at these positions. The crystal structure of T. aquaticus
region 4 with a 35 sequence element confirms the direct interaction of R584 and E585 with bases at 31 and 33, respectively. However, in the structure, DNA contact by R588 is mediated indirectly, and it is unclear how R588 provides specific base discrimination. Besides R588, many other residues within
70 region 4 also make indirect contact with the 35 region of the DNA from positions 39 to 30, primarily by interacting with the phosphate backbone, by van der Waals' contacts, or by water-mediated contacts (Campbell et al., 2002
). Thus, the surface of
70 region 4 in contact with DNA is extensive, involving two residues in region 4.1 and nine residues in region 4.2 and extending for 9 bp along the DNA.
|
|
Recognition of the TG sequence at 15/14 arises through an -helix in region 3 (Barne et al., 1997
; Campbell et al., 2002
; Sanderson et al., 2003
). [This region was originally called 2.5 (Campbell et al., 2002
).] As expected, the interaction between
70 region 4 and the
-flap is not absolutely required for transcription from
70-dependent extended 10 promoters (Kuznedelov et al., 2002
; Nickels et al., 2005
).
The T4 transcription co-activator AsiA and transcription activator MotA
AsiA is a 90 amino acid protein of T4 that is expressed early (Ouhammouch et al., 1994) and binds tightly to
70 (Stevens & Rhoton, 1975
) with a stoichiometry of 1 : 1 (Adelman et al., 1997
). Wild-type AsiA exists as a homodimer in solution (Lambert et al., 2001
; Urbauer et al., 2001
, 2002
), and the homodimer interface involves mostly hydrophobic interactions between residues in the first half of each AsiA monomer (Urbauer et al., 2002
). Although two differing AsiA structures were initially reported (Lambert et al., 2001
; Urbauer et al., 2002
), the structure of Urbauer et al. (2002)
is correct (Lambert et al., 2004a
; Urbauer et al., 2002
). AsiA was first identified as a 10 kDa factor that significantly inhibits transcription initiation from
70-dependent promoters (Stevens, 1972
, 1973
). Subsequent work assigned this protein as the product of the T4 asiA gene (Ouhammouch et al., 1994
) and demonstrated that AsiA specifically inhibits
70 recognition of the 35 DNA element, since extended 10 promoters are not inhibited by AsiA (Colland et al., 1998
; Pahari & Chatterji, 1997
; Severinova et al., 1998
). In addition, AsiA is required as a co-activator for transcription from T4 middle promoters (Hinton et al., 1996b
; Ouhammouch et al., 1995
). Thus, the binding of AsiA to
70 operates as a molecular switch that simultaneously represses transcription from host promoters, which are recognized by
70 RNA polymerase, and co-activates T4 middle promoters, which are recognized by the AsiA-bound polymerase together with the T4 transcription activator MotA. However, it appears that AsiA is not required for repression of T4 early promoters since T4 early transcription is still inhibited in a T4 asiA infection (Pene & Uzan, 2000
). In addition, in vitro work has demonstrated that transcription from several T4 early promoters is only mildly affected by the presence of AsiA (Orsini et al., 2004
).
Although AsiA is found associated with RNA polymerase holoenzyme, direct binding of AsiA to holoenyme occurs slowly, if at all (Hinton & Vuthoori, 2000). Instead, AsiA binds very readily to free
70, and it is the resulting AsiA
70 complex which then binds to core to form the AsiA-associated polymerase (Hinton & Vuthoori, 2000
). When bound to polymerase, AsiA inhibits recognition of promoters requiring a
70 contact with the 35 element by severely retarding the formation of the stable polymerasepromoter complex (Orsini et al., 2001
). However, after an extended incubation, AsiA-associated polymerase can stably bind to these promoters without the dissociation of AsiA (Orsini et al., 2001
; Pal et al., 2003
). This promoter binding in the presence of AsiA requires the DNA-binding region of the
subunits of RNA polymerase, suggesting that with a long enough incubation,
DNA contacts can eventually compensate for the lack of contacts between
70 region 4 and the 35 DNA element (Orsini et al., 2001
).
The T4 MotA protein is a transcription activator that binds as a monomer (Cicero et al., 1998; Li et al., 2002
) to the MotA box element (Brody et al., 1983
; Guild et al., 1988
; Hinton, 1991
; Schmidt & Kreuzer, 1992
) with an apparent dissociation constant of 100200 nM (Cicero et al., 1998
; Sharma et al., 1999a
). MotA also interacts with
70 (Gerber & Hinton, 1996
; Pande et al., 2002
). MotA was first identified through the isolation of T4 motA phages, which were shown to be defective in the expression of a set of T4 middle gene products (Mattson et al., 1974
, 1978
). Structural and biochemical studies have indicated that MotA contains two distinct domains: an N-terminal domain (NTD) that is formed by five
-helices and a short
-ribbon (Li et al., 2002
) and houses a transcription activation function (Finnin et al., 1997
; Gerber & Hinton, 1996
; Pande et al., 2002
) and a C-terminal domain (CTD) that by itself is capable of binding DNA (Pande et al., 2002
) and is composed of a novel double wing motif of three
-helices interspersed with six
-strands (Li et al., 2001
).
T4 middle promoters and a model for middle promoter activation
The major sequence determinants for a T4 middle promoter are a canonical 70 10 DNA element and a MotA box sequence, 5'-atTGCTTtA-3', with the C centred at position 28, 29 or 30. An analysis of more than 30 middle promoters has indicated that for most of these promoters, there is an excellent match to this consensus sequence, particularly in the core GCTT motif (Marshall et al., 1999
; Stitt & Hinton, 1994
; Truncaite et al., 2002
, 2003
). However, mutational analyses of a typical middle promoter sequence indicate that single base-pair changes within the MotA box are usually acceptable, and functional middle promoters with deviant sequences have been identified (Marshall et al., 1999
). An examination of how the loss or modification of base determinants within the MotA box sequence affects MotA binding and activation suggests that MotA uses minor groove contacts upstream and major groove contacts downstream of the centre GC, but surprisingly, it does not require any particular base feature at the highly conserved centre C·G base pair (located at position 28, 29 or 30) (Sharma et al., 1999a
). Thus, a strong match to the core GCTT motif is not an absolute requirement for MotA function. In contrast, an excellent match to the canonical
70 10 sequence is an invariant feature of middle promoters (Marshall et al., 1999
; Stitt & Hinton, 1994
; Truncaite et al., 2003
). These results have suggested that a good interaction between the
70 region 2.4 and the 10 DNA element is crucial for middle promoter usage.
Despite the requirement for MotA and AsiA in vivo, some T4 middle promoters can be recognized by polymerase alone in vitro. This is probably because middle promoters have an excellent 70 10 element and in addition, some also have the extended 10 sequence and/or a poor, but usable,
70 35 sequence element. The in vivo modification of cytosines in wild-type T4 DNA, which places a bulky glucosyl moiety in the major groove, most likely explains why RNA polymerase does not use these promoters during infection in the absence of MotA and AsiA. Such a modification should severely limit the ability of RNA polymerase to make needed major groove contacts (Sharma et al., 1999b
). Thus, RNA polymerase recognition of middle promoters in the absence of MotA and AsiA is not biologically relevant. However, this recognition has proven useful since it allows a comparison of proteinDNA contacts made by polymerase alone with those made by MotAAsiApolymerase. Such a comparison demonstrates that it is the proteinDNA contacts in the upstream promoter region that are significantly altered by the presence of MotA and AsiA (Hinton et al., 1996a
). These results have led to the idea that MotA and AsiA affect
70 contacts with the upstream promoter sequences without disturbing the typical
70 contacts with the 10 element (Hinton et al., 1996a
; Pande et al., 2002
). In this type of activation, called
appropriation, the DNA-binding CTD of MotA, rather than
70 region 4.2, contacts the upstream sequences, the MotA box, of a middle promoter (Fig. 1
, middle and bottom, right). This switch from
70 region 4.2 contact with the DNA to MotA contact with the DNA is possible because of the interaction of
70 with AsiA and with the NTD of MotA. As detailed below, the now known interactions of AsiA and MotA with residues within
70 region 4 support this model and help to explain how these interactions replace the typically extensive contacts of region 4 with DNA and with residues of the core.
appropriation is fundamentally different from the well-characterized class I or II models of prokaryotic activation, in which the tight binding of an activator with its site together with the interaction of the activator with polymerase forces
70 to contact DNA-binding elements with non-canonical
70 sequences (reviewed by Barnard et al., 2004
). To date, MotA/AsiA activation is the only known example of this type of system in prokaryotes. However, in some aspects it resembles how some eukaryotic TBP (TATA-binding protein) associated factors (TAFs) may function to change the specificity of the RNA polymerase II for different core promoter sequences (reviewed by Albright & Tjian, 2000
; Chen & Hampsey, 2002
).
Interaction of 70 region 4 with AsiA
The ability of MotA and AsiA to alter the proteinupstream DNA contacts at a T4 middle promoter while retaining the interactions of polymerase with the 10 element has suggested that one or both of the phage proteins specifically target 70 region 4. In fact, AsiA interacts with
70 residues in both regions 4.1 and 4.2 (Colland et al., 1998
; Lambert et al., 2004b
; Pahari & Chatterji, 1997
; Severinov & Muir, 1998
; Severinova et al., 1996
; Sharma et al., 1999b
; Simeonov et al., 2003
; Urbauer et al., 2001
). Two groups, Simeonov et al. (2003)
and Lambert et al. (2004b)
, have recently provided structural analyses of the AsiA
70 region 4 interaction. Both analyses indicate that the AsiA interface present in the AsiA
70 heterodimer is similar to the face that is buried in the homodimer of two AsiA proteins (Urbauer et al., 2002
). Thus, the formation of the AsiA
70 heterodimer is thought to arise through an exchange of an AsiA partner for
70 (Lambert et al., 2001
; Minakhin et al., 2001
; Urbauer et al., 2002
). However, this homodimer to heterodimer exchange may not be obligatory since AsiA bearing an N-terminal hexahistidine tag or a K20A substitution has been reported to be monomeric in solution and functional for
70 binding (Gregory et al., 2004
; Lambert et al., 2004b
).
In the NMR structure of AsiA in a complex with 70 region 4 (residues 533613) (Lambert et al., 2004b
), 18 AsiA residues, all within the N-terminal half of the protein (indicated as the red ribbon in Fig. 2
, bottom right), interact with
70 residues in regions 4.1 and 4.2 (indicated by the black lines in Fig. 2
, top). In this structure, many of the residues that
70 would normally use to contact the 35 region of DNA or to contact the core (Campbell et al., 2002
; Kuznedelov et al., 2002
; Murakami et al., 2002b
; Vassylyev et al., 2002
) interact with AsiA. In addition, the structure shows that the binding of AsiA to regions 4.1 and 4.2 dramatically reconfigures region 4 such that
70 H3-T-H4 becomes one continuous pseudohelix (Fig. 2
, bottom). As a consequence, some of the
70 residues that normally interact with DNA are positioned away from the surface, further limiting DNA contact, and portions of region 4 that would normally interact with core are also repositioned. Thus, the structure argues that AsiA inhibits in two ways: (1) it directly interacts with some of the
70 residues that normally contact the 35 element and the
-flap; and (2) it remodels region 4, creating a fundamentally different structure that lacks the architecture needed for DNA binding and the correct positioning of region 4 by core.
A previous NMR analysis (Simeonov et al., 2003) was obtained using AsiA and peptides that contain
70 region 4.1 (residues 540565) and
70 region 4.2 (residues 570599). This analysis also demonstrates that the N-terminal half of AsiA constitutes the AsiA interface in the AsiA
70 heterodimer and that the interaction of AsiA with
70 should interfere with the interaction of
70 region 4 with the
-flap. However, some findings in this NMR analysis differ from those of the AsiA
70 region 4 structure (Lambert et al., 2004b
). In the work by Simeonov et al. (2003)
, the interaction of AsiA with region 4.2 starts at residue 587, rather than residue 580 as reported by Lambert et al. (2004b)
. In addition, some of the specific
70 residues that AsiA contacts in regions 4.1 and 4.2 are not the same as those found in the structure of Lambert et al. (2004b)
. In particular, AsiA does not interact directly with
70 residues that normally contact DNA. Simeonov et al. (2003)
suggest that the ability of AsiA to inhibit the interaction of
70 with the 35 element is manifested primarily through its disruption of the normal interactions between region 4 and the
-flap, which causes a reorientation of
70 region 4 relative to core. Using luminescence resonance energy transfer, they found that the distance between
70 regions 2.4 and 4.2 is shorter in AsiA-associated polymerase than in polymerase without AsiA. This finding supports the idea that when AsiA is present,
70 region 4 is not positioned correctly for its needed interaction with the
-flap. The authors also argue that the ability of AsiA to bind to the region 4.1 or 4.2 peptides individually suggests that under certain conditions AsiA may have the flexibility to limit its interaction to just region 4.1 or 4.2.
Structural analyses are invaluable for understanding proteinprotein interactions at a molecular level. However, it is important to combine these structures with biochemical and genetic data to test the relevance of the structures to the biological system. Substantial biochemical data support many features of the AsiA70 structural analyses. The importance of some of the AsiA residues that directly contact
70 in these structures has been examined. In two investigations (Pal et al., 2003
; Sharma et al., 2002
), AsiA mutant proteins were generated both randomly and by site-specific mutagenesis. AsiA substitutions V14D, L18S, L18F and I40N, which are located within the N-terminal half of AsiA, decrease the interaction of AsiA with
70 region 4 in an E. coli two-hybrid assay (Pal et al., 2003
). Corroborating results were obtained with these AsiA mutant proteins in native protein gel assays and in transcription assays in vitro. Mutations or deletions within the 17 C-terminal residues of AsiA are not deleterious (Pal et al., 2003
; Sharma et al., 2002
). Furthermore, even a deletion of AsiA residues 4790 results in a protein that can still interact with
70 region 4 and has some ability to inhibit transcription in the absence of MotA and activate transcription in the presence of MotA (Pal et al., 2003
). In the AsiA
70 region 4 structure, the N-terminal half of AsiA contains all the residues that interact with
70, and V14, L18 and I40 are residues that make direct contacts (Lambert et al., 2004b
). In another investigation, a pull-down assay has demonstrated that an alanine substitution at D6, E10, K20, F36 or E39 disrupts the interaction between AsiA and
70 region 4 (Lambert et al., 2001
). E10, K20 and F36 also directly contact
70 in the structure. Together, these studies provide strong support for the conclusion that the AsiA interface is composed of the N-terminal half of the protein and that some of the AsiA residues that contact
70 in the structure are indeed important for this interaction.
The importance of specific 70 residues in the interaction of
70 with AsiA has also been screened in various biochemical and two-hybrid assays. Two-hybrid assays using AsiA and
70 region 4 with single substitutions within regions 4.1 or 4.2 have yielded only very modest effects, even when the
70 substitution occurs within the known binding site for AsiA (S. Pande, N. Wais, M. Vuthoori, X. B. Johnson & D. M. Hinton, unpublished). However, a modification of this assay has confirmed the importance of
70 residue F563, which contacts AsiA residues in the solution structure (Lambert et al., 2004b
) and in the NMR analysis of Simeonov et al. (2003)
. The significance of F563 was revealed using a variant of AsiA that has a K20A substitution and a variant of
70 region 4 that has a D581G substitution. The D581G change makes the helixturnhelix motif of region 4.2 (H3-T-H4 in Fig. 2
) more similar to that found in typical H-T-H DNA-binding proteins, whereas the K20 mutation is reported to result in AsiA being monomeric rather than dimeric (Gregory et al., 2004
). When used together, the AsiA K20A and
70 D581G variants interact very strongly in the two-hybrid assay, resulting in a sixfold increase in the level of the reporter gene signal over that observed with wild-type AsiA and wild-type
70 region 4 (Gregory et al., 2004
). How the AsiA K20A substitution results in a higher signal in this assay is not clear, given that this substitution disrupts the AsiA
70 interaction in a pull-down assay (Lambert et al., 2001
). Nevertheless, with such a high signal, Gregory et al. (2004)
were able to screen a set of randomly introduced mutations in
70 region 4 for ones that decrease the AsiA
70 region 4 interaction. This screen produced F563Y. In vitro transcription assays using wild-type AsiA and
70 containing only the F563Y change demonstrated that the F563Y substitution renders
70 much less susceptible to AsiA inhibition, providing independent biochemical evidence that this
70 residue is important for the
70AsiA interaction (Gregory et al., 2004
).
Structural work with polymerase holoenzyme (Murakami et al., 2002b; Vassylyev et al., 2002
) has assigned F563 as a residue that is normally involved in the
70core interaction, directly interacting with the
-flap. The interaction between the
-flap and
70 region 4 can also be observed in the two-hybrid assay provided that the
70 D581G variant is again used (Kuznedelov et al., 2002
). When using the D581G background, an F563L substitution significantly decreases the interaction of
70 with the
-flap (Gregory et al., 2004
). The targeting of F563 by AsiA is consistent with the idea that AsiA inhibits
70-dependent transcription at least in part by disrupting the contact of
70 with the
-flap. Interestingly, in the two-hybrid assays, the effects of the F563 substitutions are highly specific. The F563Y change has little effect on the
70 interaction with the
-flap while the F563L substitution does not affect
70AsiA. These results suggest that although F563 contacts AsiA, this interaction differs from its interaction with the core. Finally, combining the F563Y mutation with other mutations in region 4 that disrupt the interaction of region 4 with the
-flap renders the polymerase once again susceptible to AsiA inhibition (Gregory et al., 2004
). This result suggests that AsiA can compete with the
-flap for binding to this region of
70. Given that the binding of wild-type AsiA to wild-type
70 occurs when
70 is not bound to the core (Hinton & Vuthoori, 2000
), it may be that these
70 mutations that disrupt the
70 interaction with the
-flap now allow AsiA to access
70 when it is associated with the core. Alternatively, they may shift the dynamic equilibrium between holoenzyme and free
70 plus free core toward the free components, thus, making more free
70 available to bind to AsiA at any given time.
Recently, Gregory et al. (2005) used a different assay to identify another mutation that renders
70 less susceptible to AsiA inhibition. This assay uses a plasmid containing the
70 substitution R584A, which changes the preferred
70 35 DNA-binding element from 5'-TTGACA-3' to 5'-TTGAAA-3'. With this plasmid, one can screen for other
70 mutations in vivo that interfere with the ability of AsiA to inhibit, including mutations that would be deleterious if present in the chromosomal wild-type
70 that E. coli must use to express its genes. This assay revealed two substitutions that affect AsiA inhibition: F563Y, which was found in the earlier screen, and T552A. Transcription assays using a
70 with only the T552A substitution confirmed that this change lessens AsiA inhibition in vitro. T552 is not identified as a contact residue for AsiA in the AsiA
70 region 4 structure (Lambert et al., 2004b
) or in the earlier NMR analysis of AsiA interaction with
70 region 4 peptides (Simeonov et al., 2003
), but it is adjacent to the contact residue L551.
The effect of specific substitutions within 70 region 4.2 on its binding to AsiA have also been examined by using a series of
70 4.2 peptides, each with a single alanine substitution, in a competition assay with the wild-type 4.2 peptide (Minakhin et al., 2001
). Again, no single amino acid substitution had a dramatic effect, although substitutions at T569, V576, I590, K593, L595 and R596 were the most deleterious. In addition, polymerase with a
70 bearing either a K593E or R596E substitution required more AsiA to inhibit transcription in vitro. The AsiA
70 region 4 structural work (Lambert et al., 2004b
; Simeonov et al., 2003
) reveals a direct interaction between AsiA residues and the
70 residues I590 and L595. Although no direct interaction between T569, V576, K593 or R596 and AsiA residues is observed in these analyses, K593 and R596 lie next to contacted residues.
Interaction of 70 region 4 with MotA
The interaction of 70 with MotA is not as strong as its interaction with AsiA. Unlike AsiA, MotA is not found associated with RNA polymerase after T4 infection (Hinton et al., 1996b
), and in the E. coli two-hybrid assay, the interaction of MotA with
70 region 4 is weak, but reproducible (Pande et al., 2002
). Using this assay, Pande et al. (2002)
demonstrated that the NTD of MotA is sufficient for an interaction with
70, consistent with the idea that this portion of MotA contains an activation domain. A screen of various
70 region 4 single substitutions has yielded three, T552A, E585D and R608C, that significantly disrupt this interaction and one, E555A, that has about a twofold effect, while having little effect on the interaction of
70 region 4 with wild-type AsiA in this assay (S. Pande, N. Wais, M. Vuthoori, X. B. Johnson & D. M. Hinton, unpublished).
The far C-terminal region of 70 contains R608 and is an
-helix in the crystal structure of
region 4 of Thermotoga maritima (Lambert et al., 2004b
) (H5 in Fig. 2
). Evidence suggests that this region is crucial for the MotA
70 interaction. A substitution of the last 17 amino acid residues of
70 with those of the stationary-phase
,
s, makes multiple substitutions in this region. This
s/
70 exchange eliminates the interaction of region 4 with MotA in the two-hybrid assay, but only decreases its interaction with AsiA by about twofold (Pande et al., 2002
). In addition, the deletion of
70 residues 608613, which removes much of H5 (Fig. 2
), nearly eliminates the ability of MotA and AsiA to activate transcription from the T4 middle promoter PuvsX, but does not significantly affect the ability of AsiA to inhibit transcription from this promoter in the absence of MotA (Pande et al., 2002
).
The importance of 70 residues T552, E555 and E585 for the interaction between MotA and
70 is not yet clear. In the two-hybrid assay, replacement of either T552 or E555 with an alanine is deleterious, which is consistent with the involvement of the side chains at these residues (S. Pande, N. Wais, M. Vuthoori, X. B. Johnson & D. M. Hinton, unpublished results). Thus, there may be
70MotA contacts within regions 4.1 and 4.2, and if so, these contacts would be physically very close to those of AsiA
70. However, as yet there is no other evidence to support an involvement of these 4.1 and 4.2 residues in the interaction of MotA with
70. In addition, there is no evidence as yet to indicate that MotA and AsiA physically interact.
A genetic screen has also been employed to investigate interactions between 70 and MotA (Cicero et al., 2001
). The
70 substitutions D570N, Y571C, Y571H, L595P or S604P suppress the growth defect of a particular T4 motA mutant phage in vivo (Cicero et al., 2001
). This MotA mutant (D30A/F31A) is a positive control mutant. It is partially defective for activation of transcription but not for binding to the MotA box DNA (Finnin et al., 1997
). The identification of the suppressor S604P is consistent with an assignment of a MotA interaction site to the far C-terminal region of
70, and the identification of other suppressing residues in region 4 again suggests the possibility of other contact points for MotA in
70. However, purified
70 proteins containing each of these substitutions do not suppress the mutant MotA protein in vitro, suggesting that the in vivo results may arise from an indirect effect (Cicero et al., 2001
).
In summary, the biochemical and genetic data support an interaction between MotA and the very C-terminal region of 70 and suggest that there may be other contact points within regions 4.1 and 4.2. Two models for how both MotA and AsiA interact with
70 region 4 have been offered. Pande et al. (2002)
have proposed that the interaction between the DNA-bound MotA and the C-terminal region of
70 serves as a molecular bridge between
70 and the DNA, and thus substitutes functionally for the interaction of
70 with its 35 DNA element. MotA alone binds to a MotA box with a Kd(app) of 0·10·2 µM (Cicero et al., 1998
; Sharma et al., 1999a
), and the MotA box overlaps the 35 element for
70. Thus, in this model, the interaction of AsiA with
70 region 4 is required to promote the formation of the MotA bridge because MotA alone cannot compete with
70 effectively for the 30 to 35 region of a promoter. The AsiA
70 region 4 structure (Lambert et al., 2004b
) has features that are consistent with this idea. In this structure, the far C-terminal region of
70 is disordered, suggesting that the binding of AsiA to
70, and the ensuing conformational change, may cause a change in the C-terminal helix H5. Such a change could facilitate the connection between MotA and
70, while the interaction of AsiA with
70 regions 4.1 and 4.2 could allow MotA better access to its DNA binding site. Thus, AsiA would represent a co-activator that works by remodelling one portion of
70 so that another portion is now available for the transcriptional activator. In the other model for MotA and AsiA interaction with
70, Minakhin et al. (2003)
have proposed that the interaction of MotA with the far C-terminal region of
70 lessens AsiA binding to its contact residues in region 4.2, limiting AsiA to its 4.1 contacts. Thus, in this model, it is the interaction of AsiA with region 4.1 that causes the activation of transcription, although MotA may also make a contribution. This model is based on the finding that in the absence of MotA, AsiA activates transcription from the T4 middle promoter PrIIB2 provided that
70 lacks region 4.2 (Minakhin et al., 2003
). The finding that AsiA can bind to region 4.1 and 4.2 peptides separately supports the idea that AsiA might be able to limit its binding to just the 4.1 region (Simeonov et al., 2003
; Urbauer et al., 2001
). Despite their differences, both models share the idea that the interaction of
70 region 4 with AsiA and MotA precludes its normal interactions with the 35 element and with core.
Several class II prokaryotic activators also bind to 70 region 4. However, in many of these cases, the activator facilitates the interaction of
70 region 4 with a non-ideal 35 sequence. Investigation of the specific residues in
70 region 4.2 that are involved in the class II interaction has identified residues 588603, which are just N-terminal to H5 (Landini & Busby, 1999
; Lonetto et al., 1998
; Nickels et al., 2002
; Rhodius & Busby, 2000
). There is no indication that the far C-terminal region of
70 is needed for class II activation. Thus, the T4 system reveals another patch of
70 region 4 that can be manipulated for regulation.
Final thoughts
The recent availability of polymerase and activator structures has greatly increased our knowledge of RNA polymerase and the mechanisms of transcription initiation. As depicted in the structure-based cartoon at the bottom left of Fig. 1, it is now known that
70 interacts extensively with both the
and
' subunits of the core, and through these interactions positions its DNA-binding regions 2.4 and 4.2 so that they are in the correct spatial orientations for promoter binding. The interaction of
70 with the
-flap appears to be particularly important for the correct placement of region 4.2. In the case of activation by MotA and AsiA, structural analyses are supporting the model of
appropriation that had been developed from previous biochemical analyses. This model postulates that interactions of MotA and AsiA with
70 region 4 overcome the extensive interactions between
70 region 4 and DNA and
70 region 4 and core. Consequently, a different upstream promoter sequence can now be recognized. Although structural analyses of the MotAAsiAholoenzyme complex are not yet available, we can build a simple model based on the structure of holoenzyme and what is now known about the interactions of AsiA and MotA with
70 region 4 (bottom right of Fig. 1
). In this model the co-activator AsiA and the NTD of the activator MotA function by loosening the grip of the
-flap on region 4, thus, freeing region 4 from its normal role of contacting the DNA. The interaction of MotA NTD with
70 then positions the CTD of MotA so that it is poised for contact with the 30 MotA box. However, this remodelling of region 4 is accomplished without disturbing the needed interactions between
70 region 2.4 and the 10 region of the T4 middle promoter. Thus, this T4 system suggests that region 4 of
70 is quite flexible and has the capacity to be changed without significantly affecting the required proteinprotein and proteinDNA interactions of other
70 regions.
The MotA/AsiA example of 70 appropriation is as yet unique. However, it would be surprising if T4 middle transcription were the only case where
70 region 4 is manipulated in this way. Recent work has indicated that there is a family of proteins with similarity to AsiA in T4-type phages and bacteria (Pineda et al., 2004
). Phage orthologues have been found in the genomes of the T4-type phages RB69, 44RR, KVP40 and Aeh1, which infect a variety of Gram-negative bacteria. These phage proteins share conserved amino acids at many of the T4 AsiA residues that contact region 4 in the structure, and the KVP40 AsiA behaves like T4 AsiA in
70 binding and in transcription assays. The bacterial proteins Rsd (Jishage & Ishihama, 1998
), an anti-
70 protein of E. coli, and AlgQ (Deretic & Konyecsni, 1989
), a regulator of alginate production in Pseudomonas aeruginosa, appear to be members of this family also (Pineda et al., 2004
). Rsd and AlgQ have conserved residues at positions corresponding to T4 AsiA E10, L18, A35, F36 and V42, residues that directly contact
70 in the T4 AsiA
70 structure. Both of these proteins also interact with residues in
70 regions 4.1 and 4.2. As was observed with AsiA, the
70 substitution F563Y diminishes the Rsdregion 4 or AlgQregion 4 interaction (Pineda et al., 2004
). In addition, an R596H substitution lessens the interaction of Rsd with
70 region 4 (Dove & Hochschild, 2001
), and contact sites for Rsd in region 4.2 have been mapped to residues I590, L595 and L598 (Jishage et al., 2001
; Westblade et al., 2004
), residues that interact with AsiA in the AsiA
70 region 4 structure (Lambert et al., 2004b
). Thus, activation systems similar to that of MotA/AsiA may exist in other organisms. However, a MotA orthologue has not yet been identified in the KVP40, Aeh1, E. coli or P. aeruginosa genomes. Further work should determine the generality of the T4 system.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
Albright, S. R. & Tjian, R. (2000). TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 113.[CrossRef][Medline]
Barnard, A., Wolfe, A. & Busby, S. (2004). Regulation at complex bacterial promoters: how bacteria use different promoter organizations to produce different regulatory outcomes. Curr Opin Microbiol 7, 102108.[CrossRef][Medline]
Barne, K. A., Bown, J. A., Busby, S. J. & Minchin, S. D. (1997). Region 2.5 of the Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the extended 10 motif at promoters. EMBO J 16, 40344040.
Bown, J. A., Barne, K. A., Minchin, S. D. & Busby, S. J. W. (1997). Extended 10 promoters. In Nucleic Acids and Molecular Biology Mechanisms of Transcription, pp. 4152. Edited by F. Eckstein & D. M. J. Lilley. New York: Springer.
Brody, E., Rabussay, D. & Hall, D. (1983). Regulation of transcription of prereplicative genes. In Bacteriophage T4, pp. 174183. Edited by C. K. Mathews, E. M. Kutter, G. Mosig & P. B. Berget. Washington, DC: American Society for Microbiology.
Brody, E. N., Kassavetis, G. A., Ouhammouch, M., Sanders, G. M., Tinker, R. L. & Geiduschek, E. P. (1995). Old phage, new insights: two recently recognized mechanisms of transcriptional regulation in bacteriophage T4 development. FEMS Microbiol Lett 128, 18.[CrossRef][Medline]
Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, C. A., Weinman, O., Trester-Zedlitz, M. L. & Darst, S. A. (2002). Structure of the bacterial RNA polymerase promoter specificity sigma subunit. Mol Cell 9, 527539.[CrossRef][Medline]
Carlson, K., Raleigh, A. & Hattman, S. (1994). Restriction and modification. In Molecular Biology of Bacteriophage T4, pp. 369381. Edited by J. D. Karam and others. Washington, DC: American Society for Microbiology.
Chen, B. S. & Hampsey, M. (2002). Transcription activation: unveiling the essential nature of TFIID. Curr Biol 12, 620622.[CrossRef]
Cicero, M. P., Alexander, K. A. & Kreuzer, K. N. (1998). The MotA transcriptional activator of bacteriophage T4 binds to its specific DNA site as a monomer. Biochemistry 37, 49774984.[CrossRef][Medline]
Cicero, M. P., Sharp, M. M., Gross, C. A. & Kreuzer, K. N. (2001). Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations. J Bacteriol 183, 22892297.
Colland, F., Orsini, G., Brody, E. N., Buc, H. & Kolb, A. (1998). The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters. Mol Microbiol 27, 819829.[CrossRef][Medline]
Deretic, V. & Konyecsni, W. M. (1989). Control of mucoidy in Pseudomonas aeruginosa: transcriptional regulation of algR and identification of the second regulatory gene, algQ. J Bacteriol 171, 36803688.[Medline]
Dove, S. L. & Hochschild, A. (2001). Bacterial two-hybrid analysis of interactions between region 4 of the sigma(70) subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa. J Bacteriol 183, 64136421.
Finnin, M. S., Cicero, M. P., Davies, C., Porter, S. J., White, S. W. & Kreuzer, K. N. (1997). The activation domain of the MotA transcription factor from bacteriophage T4. EMBO J 16, 19922003.
Gardella, T., Moyle, H. & Susskind, M. M. (1989). A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity. J Mol Biol 206, 579590.[CrossRef][Medline]
Gerber, J. S. & Hinton, D. M. (1996). An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription. J Bacteriol 178, 61336139.
Gregory, B. D., Nickels, B. E., Garrity, S. J. & 7 other authors (2004). A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme. Proc Natl Acad Sci U S A 101, 45544559.
Gregory, B. D., Nickels, B. E., Darst, S. A. & Hochschild, A. (2005). An altered-specificity DNA-binding mutant of E. coli 70 facilitates the analysis of
70 function in vivo. Mol Microbiol (in press).
Gruber, T. M. & Gross, C. A. (2003). Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57, 441466.[CrossRef][Medline]
Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T. & Gold, L. (1988). Transcriptional activation of bacteriophage T4 middle promoters by the motA protein. J Mol Biol 199, 241258.[CrossRef][Medline]
Hinton, D. M. (1991). Transcription from a bacteriophage T4 middle promoter using T4 MotA protein and phage-modified RNA polymerase. J Biol Chem 266, 1803418044.
Hinton, D. M. & Vuthoori, S. (2000). Efficient inhibition of Escherichia coli RNA polymerase by the bacteriophage T4 AsiA protein requires that AsiA binds first to free sigma70. J Mol Biol 304, 731739.[Medline]
Hinton, D. M., March-Amegadzie, R., Gerber, J. S. & Sharma, M. (1996a). Characterization of pre-transcription complexes made at a bacteriophage T4 middle promoter: involvement of the T4 MotA activator and the T4 AsiA protein, a sigma 70 binding protein, in the formation of the open complex. J Mol Biol 256, 235248.[CrossRef][Medline]
Hinton, D. M., March-Amegadzie, R., Gerber, J. S. & Sharma, M. (1996b). Bacteriophage T4 middle transcription system: T4-modified RNA polymerase; AsiA, a sigma 70 binding protein; and transcriptional activator MotA. Methods Enzymol 274, 4357.[CrossRef][Medline]
Jishage, M. & Ishihama, A. (1998). A stationary phase protein in Escherichia coli with binding activity to the major sigma subunit of RNA polymerase. Proc Natl Acad Sci U S A 95, 49534958.
Jishage, M., Dasgupta, D. & Ishihama, A. (2001). Mapping of the Rsd contact site on the sigma 70 subunit of Escherichia coli RNA polymerase. J Bacteriol 183, 29522956.
Keener, J. & Nomura, M. (1993). Dominant lethal phenotype of a mutation in the 35 recognition region of Escherichia coli sigma 70. Proc Natl Acad Sci U S A 90, 17511755.
Kolesky, S. E., Ouhammouch, M. & Geiduschek, E. P. (2002). The mechanism of transcriptional activation by the topologically DNA-linked sliding clamp of bacteriophage T4. J Mol Biol 321, 767784.[CrossRef][Medline]
Kuznedelov, K., Minakhin, L., Niedziela-Majka, A., Dove, S. L., Rogulja, D., Nickels, B. E., Hochschild, A., Heyduk, T. & Severinov, K. (2002). A role for interaction of the RNA polymerase flap domain with the sigma subunit in promoter recognition. Science 295, 855857.
Lambert, L. J., Schirf, V., Demeler, B., Cadene, M. & Werner, M. H. (2001). Flipping a genetic switch by subunit exchange. EMBO J 20, 71497159.
Lambert, L. J., Schirf, V., Demeler, B., Cadene, M. & Werner, M. H. (2004a). Flipping a genetic switch by subunit exchange [correction]. EMBO J 23, 3186.
Lambert, L. J., Wei, Y., Schirf, V., Demeler, B. & Werner, M. H. (2004b). T4 AsiA blocks DNA recognition by remodeling sigma(70) region 4. EMBO J 23, 29522962.
Landini, P. & Busby, S. J. (1999). The Escherichia coli Ada protein can interact with two distinct determinants in the sigma70 subunit of RNA polymerase according to promoter architecture: identification of the target of Ada activation at the alkA promoter. J Bacteriol 181, 15241529.
Li, N., Zhang, W., White, S. W. & Kriwacki, R. W. (2001). Solution structure of the transcriptional activation domain of the bacteriophage T4 protein, MotA. Biochemistry 40, 42934302.[CrossRef][Medline]
Li, N., Sickmier, E. A., Zhang, R., Joachimiak, A. & White, S. W. (2002). The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain: the double wing motif. Mol Microbiol 43, 10791088.[CrossRef][Medline]
Lonetto, M., Gribskov, M. & Gross, C. A. (1992). The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol 174, 38433849.[Medline]
Lonetto, M. A., Rhodius, V., Lamberg, K., Kiley, P., Busby, S. & Gross, C. (1998). Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase sigma70 subunit. J Mol Biol 284, 13531365.[CrossRef][Medline]
Marshall, P., Sharma, M. & Hinton, D. M. (1999). The bacteriophage T4 transcriptional activator MotA accepts various base-pair changes within its binding sequence. J Mol Biol 285, 931944.[CrossRef][Medline]
Mattson, T., Richardson, J. & Goodin, D. (1974). Mutant of bacteriophage T4D affecting expression of many early genes. Nature 250, 4850.[Medline]
Mattson, T., Van Houwe, G. & Epstein, R. H. (1978). Isolation and characterization of conditional lethal mutations in the mot gene of bacteriophage T4. J Mol Biol 126, 551570.[CrossRef][Medline]
Mekler, V., Kortkhonjia, E., Mukhopadhyay, J. & 7 other authors (2002). Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599614.[CrossRef][Medline]
Miller, E. S., Kutter, E., Mosig, G., Arisaka, F., Kunisawa, T. & Ruger, W. (2003). Bacteriophage T4 genome. Microbiol Mol Biol Rev 67, 86156.
Minakhin, L., Camarero, J. A., Holford, M., Parker, C., Muir, T. W. & Severinov, K. (2001). Mapping the molecular interface between the sigma(70) subunit of E. coli RNA polymerase and T4 AsiA. J Mol Biol 306, 631642.[CrossRef][Medline]
Minakhin, L., Niedziela-Majka, A., Kuznedelov, K., Adelman, K., Urbauer, J. L., Heyduk, T. & Severinov, K. (2003). Interaction of T4 AsiA with its target sites in the RNA polymerase sigma70 subunit leads to distinct and opposite effects on transcription. J Mol Biol 326, 679690.[CrossRef][Medline]
Moarefi, I., Jeruzalmi, D., Turner, J., O'Donnell, M. & Kuriyan, J. (2000). Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage. J Mol Biol 296, 12151223.[CrossRef][Medline]
Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O. & Darst, S. A. (2002a). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science 296, 12851290.
Murakami, K. S., Masuda, S. & Darst, S. A. (2002b). Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296, 12801284.
Nechaev, S., Kamali-Moghaddam, M., Andre, E., Leonetti, J. P. & Geiduschek, E. P. (2004). The bacteriophage T4 late-transcription coactivator gp33 binds the flap domain of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 101, 1736517370.
Nickels, B. E., Dove, S. L., Murakami, K. S., Darst, S. A. & Hochschild, A. (2002). Protein-protein and protein-DNA interactions of sigma(70) region 4 involved in transcription activation by lambda cI. J Mol Biol 324, 1734.[CrossRef][Medline]
Nickels, B. E., Garrity, S. J., Mekler, V., Minakhin, L., Severinov, K., Ebright, R. H. & Hochschild, A. (2005). The interaction between sigma(70) and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation. Proc Natl Acad Sci U S A 102, 44884493.
Nossal, N. G. (1992). Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. FASEB J 6, 871878.
Orsini, G., Kolb, A. & Buc, H. (2001). The Escherichia coli RNA polymerase anti-sigma 70 AsiA complex utilizes alpha-carboxyl-terminal domain upstream promoter contacts to transcribe from a 10/35 promoter. J Biol Chem 276, 1981219819.
Orsini, G., Igonet, S., Pene, C., Sclavi, B., Buckle, M., Uzan, M. & Kolb, A. (2004). Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA. Mol Microbiol 52, 10131028.[CrossRef][Medline]
Ouhammouch, M., Orsini, G. & Brody, E. N. (1994). The asiA gene product of bacteriophage T4 is required for middle mode RNA synthesis. J Bacteriol 176, 39563965.[Abstract]
Ouhammouch, M., Adelman, K., Harvey, S. R., Orsini, G. & Brody, E. N. (1995). Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA polymerase to initiate transcription at T4 middle promoters. Proc Natl Acad Sci U S A 92, 14511455.
Paget, M. S. & Helmann, J. D. (2003). The sigma70 family of sigma factors. Genome Biol 4, 203 (doi:10·1186/gb-2003-4-1-203).[CrossRef][Medline]
Pahari, S. & Chatterji, D. (1997). Interaction of bacteriophage T4 AsiA protein with Escherichia coli sigma(70) and its variant. FEBS Lett 411, 6062.[CrossRef][Medline]
Pal, D., Vuthoori, M., Pande, S., Wheeler, D. & Hinton, D. M. (2003). Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma(70) subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator. J Mol Biol 325, 827841.[CrossRef][Medline]
Pande, S., Makela, A., Dove, S. L., Nickels, B. E., Hochschild, A. & Hinton, D. M. (2002). The bacteriophage T4 transcription activator MotA interacts with the far-C-terminal region of the sigma(70) subunit of Escherichia coli RNA polymerase. J Bacteriol 184, 39573964.
Pene, C. & Uzan, M. (2000). The bacteriophage T4 anti-sigma factor AsiA is not necessary for the inhibition of early promoters in vivo. Mol Microbiol 35, 11801191.[CrossRef][Medline]
Pineda, M., Gregory, B. D., Szczypinski, B., Baxter, K. R., Hochschild, A., Miller, E. S. & Hinton, D. M. (2004). A family of anti-sigma70 proteins in T4-type phages and bacteria that are similar to AsiA, a transcription inhibitor and co-activator of bacteriophage T4. J Mol Biol 344, 11831197.[CrossRef][Medline]
Rhodius, V. A. & Busby, S. J. (2000). Interactions between activating region 3 of the Escherichia coli cyclic AMP receptor protein and region 4 of the RNA polymerase sigma(70) subunit: application of suppression genetics. J Mol Biol 299, 311324.[CrossRef][Medline]
Ross, W. & Gourse, R. L. (2005). Sequence-independent upstream DNA-alphaCTD interactions strongly stimulate Escherichia coli RNA polymerase-lacUV5 promoter association. Proc Natl Acad Sci U S A 102, 291296.
Ross, W., Ernst, A. & Gourse, R. L. (2001). Fine structure of E. coli RNA polymerase-promoter interactions: alpha subunit binding to the UP element minor groove. Genes Dev 15, 491506.
Sanderson, A., Mitchell, J. E., Minchin, S. D. & Busby, S. J. (2003). Substitutions in the Escherichia coli RNA polymerase sigma70 factor that affect recognition of extended 10 elements at promoters. FEBS Lett 544, 199205.[CrossRef][Medline]
Schmidt, R. P. & Kreuzer, K. N. (1992). Purified MotA protein binds the 30 region of a bacteriophage T4 middle-mode promoter and activates transcription in vitro. J Biol Chem 267, 1139911407.
Severinov, K. & Muir, T. W. (1998). Expressed protein ligation, a novel method for studying protein-protein interactions in transcription. J Biol Chem 273, 1620516209.
Severinova, E., Severinov, K., Fenyo, D., Marr, M., Brody, E. N., Roberts, J. W., Chait, B. T. & Darst, S. A. (1996). Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. J Mol Biol 263, 637647.[CrossRef][Medline]
Severinova, E., Severinov, K. & Darst, S. A. (1998). Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA. J Mol Biol 279, 918.[CrossRef][Medline]
Sharma, M., Marshall, P. & Hinton, D. M. (1999a). Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts. J Mol Biol 290, 905915.[CrossRef][Medline]
Sharma, U. K., Ravishankar, S., Shandil, R. K., Praveen, P. V. & Balganesh, T. S. (1999b). Study of the interaction between bacteriophage T4 asiA and Escherichia coli sigma(70), using the yeast two-hybrid system: neutralization of asiA toxicity to E. coli cells by coexpression of a truncated sigma(70) fragment. J Bacteriol 181, 58555859.
Sharma, U. K., Praveen, P. V. & Balganesh, T. S. (2002). Mutational analysis of bacteriophage T4 AsiA: involvement of N- and C-terminal regions in binding to sigma(70) of Escherichia coli in vivo. Gene 295, 125134.[CrossRef][Medline]
Siegele, D. A., Hu, J. C., Walter, W. A. & Gross, C. A. (1989). Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J Mol Biol 206, 591603.[CrossRef][Medline]
Simeonov, M. F., Bieber Urbauer, R. J., Gilmore, J. M., Adelman, K., Brody, E. N., Niedziela-Majka, A., Minakhin, L., Heyduk, T. & Urbauer, J. L. (2003). Characterization of the interactions between the bacteriophage T4 AsiA protein and RNA polymerase. Biochemistry 42, 77177726.[CrossRef][Medline]
Sommer, N., Salniene, V., Gineikiene, E., Nivinskas, R. & Ruger, W. (2000). T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis. Microbiology 146, 26432653.[Medline]
Stevens, A. (1972). New small polypeptides associated with DNA-dependent RNA polymerase of Escherichia coli after infection with bacteriophage T4. Proc Natl Acad Sci U S A 69, 603607.
Stevens, A. (1973). An inhibitor of host sigma-stimulated core enzyme activity that purifies with DNA-dependent RNA polymerase of E. coli following T4 phage infection. Biochem Biophys Res Commun 54, 488493.[CrossRef][Medline]
Stevens, A. & Rhoton, J. C. (1975). Characterization of an inhibitor causing potassium chloride sensitivity of an RNA polymerase from T4 phage-infected Escherichia coli. Biochemistry 14, 50745079.[CrossRef][Medline]
Stitt, B. & Hinton, D. M. (1994). Regulation of middle-mode transcription. In Molecular Biology of Bacteriophage T4, pp. 142160. Edited by J. D. Karam and others. Washington, DC: American Society for Microbiology.
Tiemann, B., Depping, R., Gineikiene, E., Kaliniene, L., Nivinskas, R. & Ruger, W. (2004). ModA and ModB, two ADP-ribosyltransferases encoded by bacteriophage T4: catalytic properties and mutation analysis. J Bacteriol 186, 72627272.
Truncaite, L., Zajanckauskaite, A. & Nivinskas, R. (2002). Identification of two middle promoters upstream DNA ligase gene 30 of bacteriophage T4. J Mol Biol 317, 179190.[CrossRef][Medline]
Truncaite, L., Piesiniene, L., Kolesinskiene, G., Zajanckauskaite, A., Driukas, A., Klausa, V. & Nivinskas, R. (2003). Twelve new MotA-dependent middle promoters of bacteriophage T4: consensus sequence revised. J Mol Biol 327, 335346.[CrossRef][Medline]
Urbauer, J. L., Adelman, K., Urbauer, R. J., Simeonov, M. F., Gilmore, J. M., Zolkiewski, M. & Brody, E. N. (2001). Conserved regions 4.1 and 4.2 of sigma(70) constitute the recognition sites for the anti-sigma factor AsiA, and AsiA is a dimer free in solution. J Biol Chem 276, 4112841132.
Urbauer, J. L., Simeonov, M. F., Urbauer, R. J., Adelman, K., Gilmore, J. M. & Brody, E. N. (2002). Solution structure and stability of the anti-sigma factor AsiA: implications for novel functions. Proc Natl Acad Sci U S A 99, 18311835.
Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S. & Yokoyama, S. (2002). Crystal structure of a bacterial RNA polymerase holoenzyme at 2·6 Å resolution. Nature 417, 712719.[CrossRef][Medline]
Waldburger, C., Gardella, T., Wong, R. & Susskind, M. M. (1990). Changes in conserved region 2 of Escherichia coli sigma 70 affecting promoter recognition. J Mol Biol 215, 267276.[Medline]
Westblade, L. F., Ilag, L. L., Powell, A. K., Kolb, A., Robinson, C. V. & Busby, S. J. (2004). Studies of the Escherichia coli Rsd-sigma70 complex. J Mol Biol 335, 685692.[CrossRef][Medline]
Wilkens, K. & Ruger, W. (1996). Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector. Plasmid 35, 108120.[CrossRef][Medline]
Wilkens, K., Tiemann, B., Bazan, F. & Ruger, W. (1997). ADP-ribosylation and early transcription regulation by bacteriophage T4. Adv Exp Med Biol 419, 7182.[Medline]
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |