TRANSCRIPCIONES DE LAS ENTREVISTAS A LOS TUTORES DE INFANTIL.

4.1 Introduction

The sequencing of mutant amiC, amiR genes, identification of mutations and modeling of the mutations onto the AmiC structure provides limited insight into the mechanism of AmiC regulation of AmiR antitermination activity. Structural analysis of regulatory mutants would provide invaluable information into the regulatory mechanism once the three dimensional structure of AmiR is determined and the regions involved in the AmiC-AmiR interaction identified. In Chapter 3 a mutant (P A C lll) AmiC/AmiR complex was identified and isolated in the presence of butyramide. This and previous evidence make a strong case for a steric hindrance mechanism being involved in regulation of amidase expression with the AmiC/AmiR complex as the regulatory unit In order to investigate how the AmiC/AmiR complex functions in vivOy the structural interactions between AmiC and AmiR, and to gain an insight into the signal transduction mechanism which controls amidase expression, the wild-type AmiC/AmiR complex needs to be purified and crystallised. Previously (Chapter 3) pMMB66EH was used to co-overexpress mutant amiCy amiR genes in PAC452 successfully. Thus, overexpression of the wild-type amiCy amiR genes in PAC452 in the presence of butyramide should yield a stable, soluble AmiC/AmiR complex. The objective in the purifîcation was to obtain as high a yield as possible of a homogeneous wild-type AmiC/AmiR complex preparation to enable further in vitro studies and for use in crystallisation experiments.

4.2 Overexpression of the wild-type amiC, amiR

genes

Plasmid pRANl containing the andC y amiR gene fragment from PACl was mobilised into PAC452 (amiA) by triparental mating using the helper strain HB101,pRK2013 (section 2.2.1.6). The presence of pRANl in PAC452 was confirmed by conferred

resistance to carbenicillin and restriction mapping of isolated plasmid DNA with EcoRI

and Hindlll. Before attempting a large scale purification of the AmiC/AmiR complex a

small scale preparation of extracts was carried out to test whether amiC and amiR were being overexpressed by the vector and to determine the total in vivo stoichiometry of AmiC and AmiR.

4.2.1 Small scale preparation of crude whole-cell extract of PAC452,pRAN1 In the presence of butyramide

A small scale preparation of whole cell extracts from IPTG-induced and non-induced cultures of PAC452, PAC452,pMMB66EH and PAC452,pRANl was carried out as described in section 2.2.4.3 with slight modifications. Cultures were grown in the presence of 23mM butyramide and cell pellets were resuspended in Buffer B (section 2.1.4.8) + ImM PMSF. Soluble and insoluble fractions were not separated by centrifugation after sonication.

4.2.2 Analysis of cellular stoichiometry of AmIC and AmlR

Small aliquots (~ 30p,g) of total cell extracts from induced and non-induced cultures of PAC452, PAC452,pMMB66EH and PAC452,pRANl were run on a 12% SDS-PAGE gel (Figure 4.1). The induced whole cell extract of PAC452,pRANl (lane 6) shows two major new bands corresponding to AmiC and AmiR which are not present in any of the other whole cell extracts (lanes 2,3,4,5 and 7). In an attempt to determine the cellular stoichiometry of AmiC and AmiR the two bands were quantified by scanning densitometry of the stained gel.

These results confirm that the wild-type amiC and amiR genes are being overexpressed by the vector. The cellular AmiC:AmiR ratio equals 1.5:1, or 3:2 and the AmiC/AmiR complex corresponds to 9% of the total protein in the crude extract The overexpression of amiC and amiR from PAC452,pRAN 1 represents an amplifîed but ‘real’ situation with the amiC, amiR gene fragment being expressed from an external vector promoter. The AmiC/AmiR ratio shows that there is always a higher amount of AmiC compared to AmiR present in the cell, as expected for an inducible system. Furthermore, this substantiates the translational coupling mechanism proposed for amiC and amiR in Chapter 3, in which reinitiation by ribosomes occurs at less than 100%. These results

Chapter 4: RESULTS 2

3

4 5

6 7 8

9

AmiC

AmiR

F igure 4.1 SDS-PAGE analysis o f whole cell extracts o f induced and non-induced PAC452, PAC452,pMMB66EH and PAC452,pRANl.

Lane 1, MW markers; lane 2 and 3, induced and non-induced whole cell extracts o f PAC452 respectively; lanes 4 and 5, induced and non-induced whole cell extracts o f PAC452 pMMB66EH respectively; lanes 6 and 7, induced and non-induced whole cell extracts o f PAC452 pRAN 1 respectively; lanes 8 and 9, pure AmiC/AmiR complex and pure AmiC from Final Analytical Gel Filtration stage (section 4.3.6.2) respectively. The positions o f AmiC and AmiR are shown on the right.

present a scenario where, for every heterodimeric AmiC/AmiR complex produced there will be an excess of one AmiC monomer.

4.3 Purification of the wiid-type AmiC/AmiR

compiex

4.3.1 Large scale preparation of crude extract from PAC452,pRAN1

To isolate a stable wild-type AmiC/AmiR complex a crude extract of PAC452 pRANl was prepared as described in section 2.2.4.4 using Buffer A (section 2.1.4.7) + ImM PMSF. The resulting supernatant containing all soluble proteins was subjected to ammonium sulphate fractionation since this purification technique had been successfully carried out to isolate the PACl 11 AmiC/AmiR complex in Chapter 3. The initial ammonium sulphate fraction (0 to 30% saturation) was particularly useful since it removed contaminating insoluble cell debris (Chapter 3) which would otherwise block filters and FPLC columns.

4.3.2 Ammonium Sulphate fractionation

Ammonium sulphate fractionation was carried out using Buffer A + ImM PMSF as described in section 2.2.4.S to give four fractions (0 to 30%, 30 to 40%, 40 to 50% and 50 to 70% saturation). The protein concentration of each fraction was measured and typical results for total amount of protein per fraction are shown in Table 4.1. Small aliquots (l.OpL) of each fraction were analysed on a 12% SDS-PAGE gel (Figure 4.2).

Table 4.1 Total protein yield per ammonium sulphate fraction.

Ammonium sulphate fraction Total protein yield (mg)

0 to 30% saturation 2150

30 to 40% saturation 2180

40 to 50% saturation 2260

50 to 70% saturation 1460

As with the results obtained for the PAC 111 AmiC/AmiR complex (section 3.4.5), most of the wild-type AmiC/AmiR complex is present in the 40 to 50% ammonium sulphate

Chapter 4: RESULTS 2

AmiC