Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength

The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription.However,leaking transcription is common due to varied terminator strength.Besides of the representative hairpin and U-tract structure,detailed sequence and thermodynamic features of terminators were not completely clear,and the effect of terminator on the upstream gene expression was unclearly.Thus,it is still challenging to use terminator to control expression with higher precision.Here,in E.Coli,we firstly determined the effect of the 3′-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes.Secondly,terminator mutation library was constructed,and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique.The result showed that under the regulation of terminators,a negative correlation was presented between the expression of upstream and downstream genes(r=0.60),and the terminators with lower free energy corelated with higher upstream gene expression.Meanwhile,the terminator with longer stem length,more compact loop and perfect U-tract structure was benefit to the transcription termination.Finally,a terminator strength classification model was established,and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent.The results help to elucidate the role of terminators in gene expression,and the key factors identified are crucial for rational design of terminators,and the model provided a method for terminator strength prediction.

Rice Mitochondrial Genes Are Transcribed by Multiple Promoters That Are Highly Diverged

Plant mitochondrial genes are often transcribed into complex sets of mRNA. To characterize the transcription initiation and promoter structure, the transcript termini of four mitochondrial genes, atpl, atp6, cob, rps7, in rice （Oryza sativa L.）, were determined by using a modified circularized RNA reverse transcription- polymerase chain reaction method. The results revealed that three genes （atp1, atp6, rps7） were transcribed from multiple initiation sites, indicating the presence of multiple promoters. Two transcription termination sites were detected in three genes （atp6, cob, rps7）, respectively. Analysis on the promoter architecture showed that the YRTA （Y=T or C, R=A or G） motifs that are widely present in the mitochondrlal promoters of other monocot and dicot plant species were detected only in two of the 12 analyzed promoters. Our data suggest that the promoter sequences in the rice mitochondrial genome are highly diverged in comparison to those in other plants, and the YRTA motif is not an essential element for the promoter activity.

Antisense oligonucleotide technology can be used to investigate a circular but not linear RNA-mediated function for its encoded gene locus

As a class of powerful molecular tool,antisense oligonucleotides(ASOs)are not only broadly used in protein and RNA biology,but also a highly selective therapeutic strategy for many diseases.Although the concept that ASO reagents only reduce expression of the targeted gene in a post-transcriptional manner has long been established,the effect and mechanism of ASO reagents on RNA polymerase II(Pol II)transcription are largely unknown.This raised question is particularly important for the appropriate use of ASOs and the valid interpretation of ASO-mediated experiments.In this study,our results show that linear RNA ASO attenuates transcription of nascent transcripts by inducing premature transcription termination which is combinatorially controlled by Integrator,exosome,and Rat1 in Drosophila.However,circular RNA(circRNA)ASO transfection does not affect transcription activity of the encoded gene.These data suggest that the ASO technique can be applied to study a circRNA-mediated but not linear RNA-mediated function for its encoded gene locus.