Biochemical characterisation of the recombinant human 7SK snRNP

RNA Polymerase II (RNAPII) is responsible for transcribing a wide range of RNAs, including all mRNAs. After forming the pre-initiation complex on the core promoter and transcription initiation, RNAPII is known to pause just downstream of the transcriptional start site, a phenomenon known as Promoter Proximal Pausing. This pausing acts as another layer of gene expression regulation, and a number of factors can either stabilise the paused state or allow elongation to continue. Negative Elongation Factor (NELF) stabilises the paused RNAPII. Positive Transcription Elongation Factor b (P-TEFb) counteracts the effect of NELF and allows transcription to resume. P-TEFb comprises the proteins Cyclin Dependent Kinase 9 (CDK9) and Cyclin T1. P-TEFb is regulated by the 7SK small nuclear RNP, a ribonucleoprotein complex comprising the 332 nt 7SK RNA and the two core proteins Larp7 and Methylphosphate Capping Enzyme (MePCE). A HEXIM1 dimer binds to key motifs near the 5' end of 7SK, sequestering P-TEFb into the complex and inhibiting it. Upon activation by transcription factors or viral proteins, HEXIM1 and P-TEFb are released from the complex. Release of HEXIM/P-TEFb is thought to be governed by a change in 7SK secondary structure. To date, however, structural studies of the 7SK snRNP have used partial complexes reconstituted in vitro or modified 7SK RNAs, and no structure of the purified complex assembled in vivo exists. Our lab has developed a method to recombinantly express the human 7SK snRNP using baculoviral vectors. During the rotation, we have validated that the correct 7SK sequence is being expressed. The PhD project aims to biochemically characterise the complex, such as identifying post-translational modifications of the proteins and the RNA. Another area of the PhD will look at the release of P-TEFb by transcription factors using purified components or cell extracts. Finally, we propose to obtain the structure of the 7SK complex using cryogenic electron microscopy (CryoEM).