Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase.
Mobile group II introns encode a reverse transcriptase that binds the intron RNA to promote RNA splicing and intron mobility, the latter via reverse splicing of the excised intron into DNA sites, followed by reverse transcription. Previous work showed that the Lactococcus lactis Ll.LtrB intron reverse transcriptase, denoted LtrA protein, binds with high affinity to DIVa, a stem-loop structure at the beginning of the LtrA open reading frame and makes additional contacts with intron core regions that stabilize the active RNA structure for forward and reverse splicing. LtrA's binding to DIVa down-regulates its translation and is critical for initiation of reverse transcription. Here, by using high-throughput unigenic evolution analysis with a genetic assay in which LtrA binding to DIVa down-regulates translation of GFP, we identified regions at LtrA's N terminus that are required for DIVa binding. Then, by similar analysis with a reciprocal genetic assay, we confirmed that residual splicing of a mutant intron lacking DIVa does not require these N-terminal regions, but does require other reverse transcriptase (RT) and X/thumb domain regions that bind the intron core. We also show that N-terminal fragments of LtrA by themselves bind specifically to DIVa in vivo and in vitro. Our results suggest a model in which the N terminus of nascent LtrA binds DIVa of the intron RNA that encoded it and nucleates further interactions with core regions that promote RNP assembly for RNA splicing and intron mobility. Features of this model may be relevant to evolutionarily related non-long-terminal-repeat (non-LTR)-retrotransposon RTs.
Gu
Shan-Qing
SQ
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712-0159, USA.
Cui
Xiaoxia
X
Mou
Sijiong
S
Mohr
Sabine
S
Yao
Jun
J
Lambowitz
Alan M
AM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D013294:Lactococcus lactis / Q000378:metabolism; D008957:Models, Genetic; D012329:RNA, Bacterial / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018626:Retroelements
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D018626:Retroelements; C099785:LtrA protein, Lactococcus lactis; D012194:RNA-Directed DNA Polymerase