RESUMEN
At Class II CRP-dependent promoters, the Escherichia coli cyclic AMP receptor protein (CRP) activates transcription by making multiple interactions with RNA polymerase (RNAP). Two discrete surfaces of CRP, known as Activating Region 1 (AR1) and Activating Region 2 (AR2), interact with the C-terminal and N-terminal domains, respectively, of the alpha subunit of RNAP. Activating Region 3 (AR3) is a third separate surface of CRP, which is thought to interact with a target in the C-terminal region of the RNAP sigma(70) subunit. We have used a CRP mutant that functions primarily via AR3, CRP HL159 KE101 KN52, as a tool to identify residues within AR3 that are important for activation. This was achieved by screening a random mutant library of the gene encoding CRP HL159 KE101 KN52 for positive control mutants at Class II CRP-dependent promoters, and also by performing alanine scanning mutagenesis. Using both in vivo reporter assays and in vitro transcription assays, we measured the effects of key substitutions within AR3 on transcription activation in both CRP HL159 KE101 KN52 and wild-type CRP. We show that a cluster of negatively charged surface-exposed residues at positions 53, 54, 55 and 58 is required for optimal activation at a Class II, but not at a Class I, CRP-dependent promoter. We conclude that these residues in AR3 of CRP form an activatory determinant for Class II transcription activation. Abortive initiation assays were used to show that this activatory determinant accelerates the rate of isomerisation from the closed to open complex at a Class II CRP-dependent promoter. AR3 of CRP also contains an inhibitory determinant: the lysine residue at position 52 of CRP is inhibitory to maximal levels of transcription activation from Class II promoters. We show that the negative effects of K52 are not simply due to "masking" of the negatively charged residues at positions 53, 54, 55 and 58. Our results suggest that, during activation by wild-type CRP, the activatory and inhibitory determinants of AR3 balance each other. Thus, activation is predominantly determined by AR1 and AR2.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/genética , Activación Transcripcional , Alanina/genética , Sustitución de Aminoácidos/genética , Sitios de Unión , Clonación Molecular , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/genética , Genes Reporteros/genética , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Isomerismo , Cinética , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Familia de Multigenes , Mutación/genética , Regiones Promotoras Genéticas/genética , Conformación Proteica , Electricidad Estática , Relación Estructura-Actividad , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
The Escherichia coli cyclic AMP receptor protein, CRP, induces transcription at Class II CRP-dependent promoters by making three different activatory contacts with different surfaces of holo RNA polymerase. One contact surface of CRP, known as Activating Region 3 (AR3), is functional in the downstream subunit of the CRP dimer and is predicted to interact with region 4 of the RNAP sigma(70) subunit. We have previously shown that a mutant CRP derivative that activates transcription primarily via AR3, CRP HL159 KE101 KN52, requires the positively charged residues K593, K597 and R599 in sigma(70) for activation. Here, we have used the positive control substitution, EK58, to disrupt AR3-dependent activation by CRP HL159 KE101 KN52. We then screened random mutant libraries and an alanine scan library of sigma(70) for candidates that restore activation by CRP HL159 KE101 KN52 EK58. We found that changes at R596 and R599 in sigma(70) can restore activation by CRP HL159 KE101 KN52 EK58. This suggests that the side-chains of both R596 and R599 in sigma(70) clash with K58 in CRP. Maximal activation by CRP HL159 KE101 KN52 EK58 is achieved with the substitutions RE596 or RD596 in sigma(70). We propose that there are specific charge-charge interactions between E596 or D596 in sigma(70) and K58 in AR3. Thus, no increase in activation is observed in the presence of another positive control substitution, EG58 (CRP HL159 KE101 KN52 EG58). Similarly, both sigma(70) RE596 and sigma(70) RD596 can restore activation by CRP EK58 but not CRP EG58, and they both decrease activation by wild-type CRP. We suggest that E596 and D596 in sigma(70) can positively interact with K58 in AR3, thereby enhancing activation, but negatively interact with E58, thereby decreasing activation. The substitution, KA52 in AR3 increases Class II CRP-dependent activation by removing an inhibitory lysine residue. However, this increase is not observed in the presence of either sigma(70) RE596 or sigma(70) RD596. We conclude that the inhibitory side-chain, K52 in AR3, clashes with R596 in sigma(70). Finally, we show that the sigma(70) RE596 and RD596 substitutions affect CRP-dependent activation from Class II, but not Class I, promoters.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Factor sigma/química , Factor sigma/metabolismo , Supresión Genética/genética , Alanina/genética , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Secuencia Conservada/genética , Proteína Receptora de AMP Cíclico/genética , ARN Polimerasas Dirigidas por ADN/genética , Activación Enzimática , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Genes Supresores/genética , Modelos Biológicos , Modelos Moleculares , Regiones Promotoras Genéticas/genética , Unión Proteica , Conformación Proteica , Factor sigma/genética , Electricidad Estática , Transactivadores/metabolismo , Transcripción Genética/genéticaRESUMEN
We have constructed a family of promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein (CRP), with one of the sites centred between base-pairs 41 and 42 upstream from the transcription start site, and the second site located further upstream. In vivo activity measurements show that the activity of these promoters is completely dependent on CRP and that, depending on the precise location, CRP bound at the upstream site increases transcription activation. Hydroxyl radical footprinting was exploited to investigate the binding of CRP and RNA polymerase holoenzyme (RNAP) to these promoters. The study shows that the C-terminal domains of the RNAP alpha subunits bind adjacent to the upstream CRP and that their precise positioning depends on the location of upstream-bound CRP. The C-terminal domains of the RNAP alpha subunits interact with both the upstream and downstream-bound CRP via activating region 1 of CRP.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regiones Promotoras Genéticas , Secuencia de Bases , Sitios de Unión/genética , Huella de ADN , Cartilla de ADN/genética , ADN Bacteriano/química , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Conformación Proteica , Activación TranscripcionalRESUMEN
The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation.
Asunto(s)
Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Factor sigma/metabolismo , Transactivadores/metabolismo , Alanina , Secuencia de Aminoácidos , Factor de Transcripción de AraC , Proteínas Bacterianas/genética , Sitios de Unión , Secuencia Conservada , Proteína Receptora de AMP Cíclico/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Secuencias Hélice-Giro-Hélice , Proteínas Hierro-Azufre/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/metabolismo , Factor sigma/química , Factor sigma/genética , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Most bacterial transcription activators function by making direct contact with RNA polymerase at target promoters. Some activators contact the carboxy-terminal domain of the RNA polymerase alpha subunit, some contact region 4 of the sigma70 subunit, whilst others interact with other contact sites. A number of activators are ambidextrous and can, apparently simultaneously, contact more than one target site on RNA polymerase. Expression from many promoters is co-dependent on two or more activators. There are several different mechanisms for coupling promoter activity to more than one activator: in one such mechanism, the different activators make independent contacts with different target sites on RNA polymerase.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Transactivadores/metabolismo , Activación Transcripcional , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Regiones Promotoras GenéticasRESUMEN
Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at Class II promoters is dependent on direct interactions between two surface-exposed activating regions (AR1 and AR2) and two contact sites in RNA polymerase. The effects on transcription activation of disrupting either AR1 or AR2 have been measured at different Class II promoters. AR2 but not AR1 is essential for activation at all the Class II promoters that were tested. The effects of single positive control substitutions in AR1 and AR2 vary from one promoter to another: the effects of the different substitutions are contingent on the -35 hexamer sequence. Abortive initiation assays have been used to quantify the effects of positive control substitutions in each activating region on the kinetics of transcription initiation at the Class II CRP- dependent promoter pmelRcon. At this promoter, the HL159 substitution in AR1 results in a defect in the initial binding of RNA polymerase whilst the KE101 substitution in AR2 reduces the rate of isomerization from the closed to the open complex.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/química , Regiones Promotoras Genéticas/genética , Activación Transcripcional/genética , Secuencia de Bases , Sitios de Unión/genética , Proteínas Portadoras , Clonación Molecular , Proteína Receptora de AMP Cíclico/farmacología , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Cinética , Datos de Secuencia Molecular , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The Escherichia coli cyclic AMP receptor protein (CRP) is a homodimeric transcription activator triggered by cyclic AMP. Escherichia coli contains more than 100 different promoters that can be activated by CRP: in most cases the CRP acts by making direct contact with RNA polymerase. Remarkably, there is considerable variation in the location of the DNA site for CRP from one CRP-dependent promoter to another. Genetic methods have been used to locate the activating regions of CRP that make contact with RNA polymerase at promoters of different architectures. At promoters where the DNA site for CRP is centred near to positions -61, -71 or -81 (i.e. 61, 71 or 81 base pairs upstream of the transcript start-point, respectively), a single surface-exposed loop (Activating Region 1) in the downstream subunit of the CRP dimer makes contact with RNA polymerase. The contact site in RNA polymerase is located in one of the C-terminal domains of two RNA polymerase alpha subunits. At promoters where the DNA site for CRP is centred near to position-41, both subunits of the CRP dimer make contact with RNA polymerase via three separate surface exposed regions (Activating Regions 1, 2 and 3). At these promoters, where bound CRP overlaps with RNA polymerase-binding elements, the C-terminal domains of the polymerase alpha subunits are displaced and bind upstream of CRP. Activation at a number of E. coli promoters is dependent on binding of two CRP dimers, with one dimer bound near to position-41 and the other dimer bound further upstream. In these cases, both bound CRP dimers contact RNA polymerase. The CRP dimer bound around position-41 contacts RNA polymerase via Activating Regions 1, 2 and 3, whereas the upstream bound CRP dimer contacts one of the displaced alpha C-terminal domains via Activating Region 1 in the downstream CRP subunit. Thus in these cases, codependence on two activators is due to simultaneous contacts between separate activators and RNA polymerase. This mechanism allows great flexibility, as any activator that can contact the C-terminal domain of the RNA polymerase alpha subunits can act cooperatively with CRP.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regiones Promotoras Genéticas , Transactivadores/química , Transactivadores/metabolismo , Activación Transcripcional , Sitios de Unión , Sustancias Macromoleculares , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region. Transcription activation at class II CRP- dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96. These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters. Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regiones Promotoras Genéticas , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión/genética , Proteínas Portadoras , Proteína Receptora de AMP Cíclico/química , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Bacterianos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Activación TranscripcionalRESUMEN
Transcription activation at two semi-synthetic Escherichia coli promoters, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream from the respective transcription startpoints. An UP-element that can bind the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit was cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5). In both cases CRP-dependent promoter activity was increased by the UP-element, but CRP-independent activity was not increased. DNase I footprinting was exploited to investigate the juxtaposition of bound CRP and RNAP alpha-subunits. In both cases, CRP and RNAP alpha-subunits occupy their cognate binding sites in ternary CRP-RNAP promoter complexes. RNAP alpha-subunits can occupy the UP-element in the absence of CRP, but this is not sufficient for open complex formation. The positive effects of binding RNAP alpha-subunits upstream of the DNA site for CRP at -41.5 are suppressed if the UP-element is incorrectly positioned.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Secuencia de Bases , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
The effects of a number of mutations in crp have been measured at different cyclic AMP receptor protein (CRP)-dependent Class II promoters, where the CRP-binding site is centred around 41 1/2 base pairs upstream from the transcription start point. The amino acid substitutions HL159 and TA158 result in reduced CRP-dependent activation, but the reduction varies from one Class II promoter to another. Deletions in the C-terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158. The role of the C-terminus of alpha at these promoters is assessed. Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is discussed.