Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3.

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.

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.

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.

Transcription activation at promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organisation of the RNA polymerase alpha subunits.

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.

Positive activation of gene expression.

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.

Transcription activation at class II CRP-dependent promoters: the role of different activating regions.

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.

Orientation of functional activating regions in the Escherichia coli CRP protein during transcription activation at class II promoters.

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.

Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit.

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.