Mechanisms Of Ribosome Stalling By SecM At Multiple Elongation Steps

In central dogma,genetic information delivery from mRNA into protein,is performed by the large molecular machine ribosome.Ribosome itself acts as a hub in protein quality control processes to regulate nascent peptides' fate,by proofreading protein biogenesis and dowmstream gene initiating.During ribosomal translation,a series of protein binding factors work on ribosome inducing ribosome synthesizes protein at a nonconstant rate,modifying and helping nascent polypeptide correct folding,to participate in protein quality control.Accumulating evidences have revealed that nascent polypeptide chain can be stalled in the ribosome exit tunnel during its synthesis,interacting with ribosomal components to regulate the expression of co-transcribed genes.In Escherichia coli,SecM is a secretory protein(170 amino acids),that its C terminual sequences mediate ribosomal translation stalling and initiate downstream gene expression.Biochemical and structural researches indicate that the 17-amnio-acid stalling sequence(150FSTPVWISQAQGIRAGP166)near its C terminus will interact with 23 S rRNA,ribosome protein uL4 and uL22,and stay in the exit tunnel with a compact conformation,which will inactive the peptidyl-transferase center(PTC)and lead stalling.However,the mechanism of SecM stalling is still unknown at atomic level.What's more,increasing evidences show that the stalling may occure in a dynamic process.Therefore,we use cryo-EM single particle technique to study the mechanism of SecM stalling,and get two cryo-EM structures of secM-ribosome complex at a resolution of 3.5~3.7?.With biochemical and structural analysis,we propose that SecM stalls ribosome translation by two different mechanisms and at different translation elongation stage.One is stalling ribosome at an unrotated state,inactiving the PTC center and going against the accommodation of the incoming aminoacyl-t RNA to the A site.The other is stalling ribosome at a rotated state,elongating the process of nascent peptidyl-RNA translocating from the hybrid A/P site to P/P site.R163,one amino acid of the SecM stalling sequence,is crucial in both of these two mechanisms.With the help of the other stalling amino acids,R163 locates at the specific site in the exit tunnel and interacts with ribosomal components,inducing 50 S comformational change to disfavor translation elongation.In conclusion,we demonstrate for the first time that there are two major translational stalling mechanisms by SecM,revealing the details how SecM interacts with ribosome and mediates translation elongation at a continuous and multiple steps.Taking together with previous reported studies,including antibiotics and leader peptides,it indicates that nascent polypeptides and small ligands can mediate ribosome conformational change and fine-tune translation rate with different mechanisms.