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Despite optimisation, microseeding and additive screening, the conditions permissive for the growth of crystals with high resolution diffraction remained elusive. A possible explanation for this is intrinsic flexibility of YonO during crystallisation. RNAPs contain mobile domains. In the multisubunit RNAP, the clamp domain swings down to grip the downstream DNA (Chakraborty et al., 2012, Cramer et al., 2000, Cramer et al., 2001). The notion that YonO contains similarly mobile domains is plausible, as it was observed that QDE-1, a distant homologue of YonO, is able to adopt open and closed

conformations through movement of the head and slab domains (Salgado et al., 2006)(Figure 7-6).

Figure 7-6: Open and closed conformation of QDE-1. Taken from Salgado et al, 2006. The side view of a QDE-1 monomer is shown. The slab and head domains of two monomers belonging to the same homodimer were seen to be positioned at different angles. This is represented by double headed arrows between monomer A and

monomer B. In an independent, second dimer structure, the head and slab domains are positioned at the same angle. Taken together this suggests the head and slab domains have a degree of flexibility which may correspond to the opening and closing of QDE-1.

Whilst the mobility of RNAP domains was not problematic for the growth of well ordered, high quality crystals previously, it cannot be ruled out that potential intrinsic

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flexibility of YonO is detrimental to crystal formation. To address this potential reason for poor crystal diffraction, we sought to crystallise YonO bound to a nucleic acid scaffold. It was hoped that upon binding to the scaffold, YonO would adopt a stable, rigid conformation which would allow for increased order within the crystal. For the determination of the Thermus thermophilus elongation complex structure, RNAP was assembled with a nucleic acid scaffold prior to crystallisation (Vassylyev et al., 2007a). This scaffold (denoted as scaffold 1) was obtained and used for the

assembly of YonO elongation complexes (YonO EC) (scaffold 1, Figure 7-7). To confirm that YonO ECs were both stable and active, assembled complexes were immobilised by the 5’ biotin tagged template DNA strand to streptavidin beads and washed with transcription buffer containing low or high concentrations of salt. Only stable elongation complexes are resistant to high ionic strength (Sidorenkov et al., 1998). Upon washing the immobilised complexes with high salt transcription buffer, non- template DNA (NTDNA14) and RNA (RNA16) was lost, showing the YonO EC were unstable (Figure 7-7, compare lane 1 against lane 2 and lane 6 against lane 7). The assembled complexes were not destabilised by low salt concentrations, as RNA16 and NTDNA14 were not removed after washing immobilised complexes with 40 mM KCl. Furthermore, complexes extended RNA16 to the end of the template in the presence of NTPs (Figure 7-7, compare lane 4 against lane 5 and lane 9 against 10,). However, not all YonO elongation complexes extended RNA16. Scaffold 1 was originally designed for form stable T. thermophilus elongation complexes that do not oscillate between the post and pre-translocated states, which would interfere with crystallisation. Therefore, the inability of all YonO complexes to extend RNA16 in the presence of NTPs can be explained by increased stability of complexes due to the sequence of scaffold 1 oligonucleotides (Vassylyev et al., 2007a).

Scaffold 1 was comprised of the minimum nucleic acid strands required to form a stable elongation complex. The short length of the scaffold was designed to reduce the likelihood that either RNA or DNA extrudes from the surface of RNAP and interferes with crystal contacts during crystallisation (Kashkina et al., 2006). Whilst bacterial RNAP is able to form stable elongation complexes on this scaffold, YonO did not. The non-template DNA strand is known to have a stabilising effect on elongation

(NTDNA14) was short, with the strand only annealing to the downstream region of the template DNA strand (as seen in Figure 7-7). It was reasoned that a longer non-

template strand may increase the stability of YonO elongation complexes (referred to as scaffold 2 which is shown in Figure 7-7). The non-template strand was extended at the 5’ end by 9 nucleotides (NTDNA23). The extended sequence was non-

complementary to the template strand to mimic the transcription bubble (Yin and Steitz, 2002). Scaffold 2 YonO ECs were assembled, immobilised and washed with transcription buffer containing high or low concentrations of salt. The stability of the complexes was improved by the presence of the longer non-template DNA strand as both RNA13 and NTDNA23 were present after washing scaffold 2 complexes with high salt buffer (Figure 7-7, compare lanes 11 and 17 to lanes 1 and 7,). The increased stability of scaffold 2 EC is further demonstrated by comparisons of RNA13 extension by scaffold 1 and 2 complexes radiolabelled at the 5’ end of the non-template strand (Figure 7-7, compare lane 18 to 8,). In these reactions, RNA13 extended by YonO was labelled through the incorporation of α-[32P]-AMP during elongation. Scaffold 2 resulted in increased amounts of extended RNA13 which suggests more RNA13 was retained in assembled elongation complexes after washing with high salt buffer. One complication with the crystallisation of YonO is the requirement of 500 mM NaCl to maintain solubility of YonO at a concentration of 10 mg ml-1 (not discussed or shown). When determining the stability of scaffold 1 and 2 elongation complexes, complexes were formed in the presence of 40 mM KCl prior to being washed by 500 mM NaCl transcription buffer. This differs to the formation of elongation complexes intended for crystallisation screens, which would be formed in the presence of the 500 mM NaCl necessary for YonO solubility. Elongation complexes are only resistant to high ionic strength after assembly, making it possible that elongation complexes may not form in 500 mM NaCl prior to crystallisation trials (Sidorenkov et al., 1998). Nevertheless, since scaffold 2 complexes showed increased stability at high salt concentrations, scaffold 2 was incubated with YonO prior to overnight equilibration against the standard set of crystallisation screens (see materials and methods). The screens resulted in bipyramidal crystals, all of which, when harvested and subjected to synchrotron radiation, diffracted to 7 – 8 Å. It is likely that the salt concentration prevented elongation complex formation as predicted and that the crystals did not

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include the nucleic acid scaffold. YonO appears to not be like other RNAPs, which require a much reduced concentration of salt to remain soluble at 10 mg ml-1 allowing EC formation at the high protein concentrations required for crystallisation (Gnatt et al., 2001, Kashkina et al., 2006, Vassylyev et al., 2007a).

To attempt to reduce the salt concentration required for YonO solubility, 10 mg ml-1 YonO in 500 mM NaCl buffer was incubated with scaffold 2 before undergoing serial dialysis against buffers containing decreasing concentrations of salt. It was hoped the presence of a nucleic acid scaffold would alter the solubility of YonO and allow high protein concentrations in reduced salt concentrations. This was not the case, as with each dialysis against a lower salt concentration, precipitation occurred.

For the crystallisation of RNAP II elongation complexes, RNAP II was allowed to form elongation complexes, either through incubation with a scaffold or by transcribing on duplex DNA with a 3’ single stranded tail. RNAP II started transcription on the tail which bypassed the need for initiation factors. The sequence of the downstream DNA permitted synthesis of a 14 nucleotide RNA without incorporation of UMP. When provided all NTPs except UTP, all active RNAP II complexes paused upon reaching the first Adenosine in the template DNA ensuring all RNAP II complexes were

homogenous. (Kettenberger et al., 2004, Gnatt et al., 2001). In both methods,

elongation complexes were purified and concentrated prior to crystallisation screens. Whilst not attempted due to time constraints, one of these approaches may allow for YonO elongation complexes to be maintained in high salt conditions. After initial assembly in low salt and low protein concentrations, elongation complexes would be purified by reverse heparin chromatography or gel filtration. Subsequently YonO elongation complexes would be concentrated to 10 mg ml-1 and trialled for crystallisation.

Figure 7-7: Determination of optimum nucleic acid scaffold for YonO elongation complex crystallisation. YonO elongation complex stability and activity with three different nucleic acid scaffolds was determined. Scaffold sequences were based on that used for the crystallisation of the bacterial elongation complex (scaffold 1) (Vassylyev et al., 2007a). Scaffolds are numbered, with sequences below. The scaffold schematic indicates the

position of 32P labelling (denoted by *). RNA, template DNA and non-template DNA are coloured red, black and blue, respectively. Elongation complexes were assembled and washed with a high (500 mM NaCl) or low (40 mM KCl) concentration of salt. Transcription was initiated by the addition of 100 µM NTPs. RNA in complexes labelled on the non-template DNA strand was visualised by the incorporation of α-[32P]-ATP in the presence of 10 µM ATP and 100 µM GTP, CTP and UTP. Reactions were incubated at 37 ⁰C for 30 minutes before being terminated by the addition of stop buffer and resolved by denaturing PAGE.

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