Non-coding RNA Participates Core Phage And Bacteria Functions Via Conformational Changes

Non-coding RNA?ncRNA?transcribed from the genome generally does not encode proteins.It participates a variety of life activities,such as evolution,metabolism,embryogenesis and viral infection.The incredibly diverse functions and regulatory patterns of non-coding RNA?ncRNA?endorse them as the centerpiece of current research attention.RNA's diverse structure and dynamics provides its molecular functions.Some ncRNAs form alternative base-pairing with multiple con-formations.Therefore,in order to study the function of ncRNA,we need to analyze the structural characteristics of the RNA element,so as to grasp the conformational changes in the process and speculate its mechanism.Currently,high-throughput sequencing and bioinformatics technologies have discovered many new non-coding RNAs,yet providing little insight towards their mechanisms.These new non-coding RNAs generally falls into two categories based on function:structural-func-tional non-coding RNAs and regulatory non-coding RNAs.This study employs a combination of crystallography,small angle scattering and chemical probing techniques.We chose the phi29 phage:prohead RNA?pRNA?and Escherichia coli:yybP riboswitch as model systems to represent both categories.We investigated the conformational changes of the pRNA-gp16 RNP during ATP hy-drolysis,indicating that pRNA domain II has a very important physiological function and can par-ticipate in the DNA packaging process.Meanwhile,comparing the E.coli yybP riboswitch dynamic with or without Mn²⁺in different pH,we characterize the yybP riboswitch as a tandem riboswitch which is simultaneously regulated by Mn²⁺concentration and pH.This study lays the foundation for studying non-coding RNA's role in double-stranded DNA viral packaging process and the regulatory role of bacteria in coping with environmental stress.Structural-functional non-coding RNA?pRNA?changes conformation in the viral ge-nome packaging process.The non-coding pRNA and the ATPase gp16 are important transport components in the phi29 DNA packaging motor.The gp16 drives for DNA packaging process by hydrolyzing ATP.The highly conserved pRNA serves as the main structural hub between the gp16and the viral capsid.Understanding how pRNA interacts with gp16 will clarify the molecular mech-anisms of packaging motors.We solved the crystal structure of pRNA domain II and presented the beads model of pRNA-gp16 complex and its conformational changes occur with ATP or ADP bind-ing.We find that gp16 specifically binds to pRNA domain II and pRNA-gp16 forms a"Z"-shaped complex.With ATP binding,the pRNA-gp16 complex forms a“closed”motor.The pRNA domain II sequentially opens during ATP hydrolysis,showing an"Open"state,and finally pRNA-gp16 com-plex resets after ADP releasing.This study suggests that pRNA changes conformation during DNA packaging process.pRNA domain II can close or open the dsDNA channel in response to ATP or ADP binding.Our results indicate that pRNA domain II actively participates in the DNA packaging process.Tandem regulation mechanism of regulated non-coding RNA.Tandem Riboswitch controls essential biological pathways responding to multiple stimuli,similar to the Lac operon in bacteria.The yybP-ykoY motif constitutes the most common riboswitch classes.However,its structure,con-formational change and dynamics remained uncharacterized.Sequence analysis revealed a novel pH-responsive RNA element containing yybP-ykoY consensus sequence in the 5?untranslated region of alx gene in E coli.Here we solved the crystal structure of yybP-ykoY motif and characterized it as a Mn²⁺-dependent yybP riboswitch.Its Mn²⁺-binding core domain shares similarities with the published lactococcus lactis yybP-ykoY riboswitch structure,implying these two Mn²⁺-dependent riboswitches utilize a highly conserved ligand-recognition mechanism.Then we performed small angle X-ray scattering?SAXS?on pH-responsive element which contains yybP riboswitch aptamer domain and expression platform at different pH conditions,with or without Mn²⁺.We observed multiple conformations upon Mn²⁺binding under various pH conditions?pH=6.0⁸.0?.The above results indicate that the yybP riboswitch in E.coli can be simultaneously affected by the concentra-tion of Mn²⁺and pH condition,synergistically regulating the expression of downstream alx genes.In this dissertation,a variety of structural biological methods have demonstrated complimen-tary advantages during the non-coding RNA characterization.By analyzing the structural data of these non-coding RNA elements,we observed and detected dynamic conformational changes and concluded the important molecular roles of pRNA in the DNA packaging process and the tandem regulation mechanism of the yybP riboswitch.