Co-transcriptional Folding Mechanism For Ribozyme And Riboswitch

HDV ribozyme is a 1.7kb genome.The ribozyme region of the RNA molecule is about 85-nt.Co-infection of HDV and HBV for human being will exacerbate the severity of disease.The ribozyme performs the self-cleavage activity through folding to a double pseudoknot structure.Flanking regions often could affect the co-transcriptional folding behavior.Because the functional structure of the ribozyme is a pseudoknot,hence,the pseudoknots will be introduced to our master equation with time and to study the effects of flanking regions on HDV ribozyme co-transcriptional folding kinetics.Candida intron is a 379-nt RNA sequence and a group I intron,which can performs the self-splicing activity.The diseases for the intron are mainly caused by canidia Albicans with a chronic,subacute or acute infection.Besides,the intron invades the skin and mucosa of the human body and also causes visceral and systemic infectious diseases.Candida intron can be separated from its precursor RNA molecule by two transesterification reactions.Simialr with HDV ribozyme,the functional structure of Candida intron is a complex P3-P7 pseudokont.Because the intron is too long and the structures are too complex so that we studied the transcriptional folding kinetics for Candida intron by using a piecewise extended method of master equation with time.The secondary structure of c-di-AMP bound ydaO riboswitch comprises two three-way junctions connected by two helices and a large conserved interior loop,so,the ydaO riboswitch can sense two c-di-AMP molecules.Furthermore,a long range pseudoknot,which is formed by pairing of the middle and 3' terminal regions,closes the domain.Hence,the ydaO riboswitch is quite different with many other riboswitches.In addition,the biological function of the riboswitch is ususlly determined by the structure formed during the transcription process.Therefore,revealing how riboswitch switches into different functional structures at different transcriptional conditions,such as ligand concentration,transcription rate and transcriptional pause,is the key to study the regulation of gene expression by riboswitches.Here,the mechanism of ligand binding kinetics is introduced into our model.The main results of this paper are as follows:(1)A long-chain RNA transcriptional folding kinetics method containing pseudoknot structures was established.This method can be used to predict the structure and folding path of long-chain RNA containing pseudoknot structures under different transcription rates,transcriptional pauses and so on.(2)The effects of RNA sequences from different regions on the transcriptional folding process of HDV ribozymes were predicted theoretically.The commonality of the ribozyme region and the specificity of the upstream and downstream regions on the self-cleavaged structure of HDV ribozyme during HDV ribozyme transcriptional folding were revealed.For all the sequences,the ribozyme regions(from 1 to 73)all fold to the same structure during transcription.The 99 nt HDV is quite different with its refolding kinetics.The existence of the 30 nt upstream flanking sequence can inhibit the ribozyme region folding into the active native state through forming a helix Alt1 with the segment 70-90.The longer upstream flanking sequence 54 nt itself forms a stable hairpin structure,that sequesters the formation of Alt1 helix and leads to rapid formation of cleavage-active structure.Although the 55 nt downstream flanking sequence could invade the already folded active structure during transcription by forming a more stable helix with the ribozyme region,the slow transition rate could keep the structure in the cleavage-active structure to perform the activity.(3)The kinetics of transcriptional folding of Candida intron was theoretically predicted,revealing the importance of the first 11 nt sequence fragment to the self-splicing active structure for Candida intron.The native sequence of the Candida intron has two folding paths,one foldes to the active P3-P7 structure and the other foldes to the stable intermediate structure.The 11 nt Candida was only one major path and was directly foldes to the active P3-P7 structure.(4)The mechanism of ligand binding kinetics was introduced,and this mechanism was combined with the mechanism of transcriptional folding dynamics of RNA containing pseudoknot to theoretically elaborate the regulation mechanism of ydaO riboswitch,which is is the thermodynamic and kinetic regulation mechanism.Furthermore,the effects of c-di-AMP ligand concentration,transcription rate,ligand dissociation rate and binding rate on the regulation of ydaO riboswitches were given.Higher ligand concentrations,higher transcription rates,and larger ligand binding rates can lead to more structures convert to the gene expression OFF state;lower ligand concentrations or lower transcription rates can lead to more the structures transition to the gene expression ON state.