Analysis of interactions between Activating Region 1 of Escherichia coli FNR protein and the C-terminal domain of the RNA polymerase alpha subunit: use of alanine scanning and suppression genetics.

Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) during transcription activation at FNR-regulated promoters. Using an alphaCTD alanine scan mutant library, we have identified the residues of alphaCTD that are important for FNR-dependent transcription activation. Residues Asp-305, Gly-315, Arg-317, Leu-318 and Asp-319 are proposed to be the key residues in the contact site on alphaCTD for Activating Region 1 of FNR. In previous work, it had been shown that Activating Region 1 of FNR is a large surface-exposed patch and that the two crucial amino acid residues are Thr-118 and Ser-187. In this work, we have constructed Arg-118 FNR and Arg-187 FNR and shown that both FNR derivatives are defective in transcription activation. However, the activity of FNR carrying Arg-118 can be partially restored by substitutions of Lys-304 in alphaCTD. Similarly, the activity of FNR carrying Arg-187 can be partially restored by substitutions of Arg-317 or Leu-318 in alphaCTD. The specificity of the restoration suggests that, during transcription activation by FNR, the side-chain of residue 118 in Activating Region 1 of FNR is located close to Lys-304 and Asp-305 in alphaCTD. Similarly, the side-chain of residue 187 in Activating Region 1 of FNR is located close to Arg-317 and Leu-318 in alphaCTD. These results can be used to model the interface between Activating Region 1 of FNR and its contact target in alphaCTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcription activator, CRP and alphaCTD.

Role of activating region 1 of Escherichia coli FNR protein in transcription activation at class II promoters.

FNR is an Escherichia coli transcription factor that activates gene expression in response to anaerobiosis at a large number of promoters by making direct contacts with RNA polymerase. At class II FNR-dependent promoters, where the DNA site for FNR overlaps the -35 element, activating region 1 of FNR is proposed to interact with the C-terminal domain of the RNA polymerase alpha-subunit. Using a model class II FNR-dependent promoter, FF(-41.5), we have performed in vivo and in vitro experiments to investigate the role of this interaction. Our results show that FNR, carrying substitutions in activating region 1, is compromised in its ability to promote open complex formation and thus to activate transcription. Abortive initiation assays were used to assess the contribution of activating region 1 of FNR to open complex formation. A new method for the purification of the FNR protein is also described.

Repression of transcription initiation by Escherichia coli FNR protein: repression by FNR can be simple.

Naturally occurring promoters that are repressed by the Escherichia coli FNR protein are complex. In this work, we have constructed a simple semi-synthetic promoter that is repressed by FNR binding to a single site that overlaps the promoter -35 element. Our results show that a single site for FNR is sufficient for effective repression. This semi-synthetic promoter provides a simple tool for monitoring FNR binding to target sites in the absence of its activation function. We have exploited this to study FNR mutants that are defective in repressing the ndh promoter, a complex naturally occurring promoter that is repressed by FNR.

Transcription activation at class I FNR-dependent promoters: identification of the activating surface of FNR and the corresponding contact site in the C-terminal domain of the RNA polymerase alpha subunit.

A library of random mutations in the Escherichia coli fnr gene has been screened to identify positive control mutants of FNR that are defective in transcription activation at Class I promoters. Single amino acid substitutions at D43, R72, S73, T118, M120, F181, F186, S187 and F191 identify a surface of FNR that is essential for activation which, presumably, makes contact with the C-terminal domain of the RNA polymerase alpha subunit. This surface is larger than the corresponding activating surface of the related transcription activator, CRP. To identify the contact surface in the C-terminal domain of the RNA polymerase alpha subunit, a library of mutations in the rpoA gene was screened for alpha mutants that interfered with transcription activation at Class I FNR-dependent promoters. Activation was reduced by deletions of the alpha C-terminal domain, by substitutions known to affect DNA binding by alpha, by substitutions at E261 and by substitutions at L300, E302, D305, A308, G315 and R317 that appear to identify contact surfaces of alpha that are likely to make contact with FNR at Class I promoters. Again, this surface differs from the surface used by CRP at Class I CRP-dependent promoters.

Spacing requirements for transcription activation by Escherichia coli FNR protein.

We cloned a consensus DNA site for the Escherichia coli FNR protein at different locations upstream of the E. coli melR promoter. FNR can activate transcription initiation at the melR promoter when the FNR binding site is centered around 41, 61, 71, 82, and 92 bp upstream from the transcription start. The SF73 positive control amino acid substitution in FNR interfered with transcription activation by FNR in each case. In contrast, the GA85 positive control substitution reduced activation only at the promoter, where the FNR binding site is 41 bp upstream of the transcript start. The SF73 substitution appears to identify an activating region of FNR that is important for transcription activation at promoters that differ in architecture. Experiments with oriented heterodimers showed that this activating region is functional in the upstream subunit of the FNR dimer at the promoter where FNR binds around 41 bp from the transcript start and in the downstream subunit at the promoters where FNR binds farther upstream.

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.

Cranial sixth-nerve palsy and eosinophilia in an outbreak of Mycoplasma pneumonia.

The authors discuss a case in which three siblings presented with Mycoplasma pneumonia. All three had a typical rise in complement fixation antibody titres. However, the sibling with the highest titre also developed cranial sixth-nerve palsy; in addition, she was the only one of the three who did not have an eosinophilia. The authors review the symptomatology of Mycoplasma pneumonia and the involvement of the central nervous system.

A first mathematical approximation of cerebral cortical responses to cerebello-thalamic signals using a thermodynamic analogue.

A mathematical approximation is developed to predict cerebral cortical responses CTX(t, x, R) at a distance R (R greater than or equal to 0) from a focal point in the cerebral cortex, at a depth x (0 less than or equal to x less than or equal to 500 mu) from the cortical surface and at any time t, to thalamic signals THAL(t) whose amplitudes also vary with time t. The equation is derived from a thermodynamic analogue, which is the time dependent heating of a focus at the surface of a semi-infinite solid.