PMID- 35713547
OWN - NLM
STAT- MEDLINE
DCOM- 20221219
LR  - 20221222
IS  - 1362-4962 (Electronic)
IS  - 0305-1048 (Print)
IS  - 0305-1048 (Linking)
VI  - 50
IP  - 12
DP  - 2022 Jul 8
TI  - Direct tracking of reverse-transcriptase speed and template sensitivity: 
      implications for sequencing and analysis of long RNA molecules.
PG  - 6980-6989
LID - 10.1093/nar/gkac518 [doi]
AB  - Although reverse-transcriptase (RT) enzymes are critical reagents for research 
      and biotechnology, their mechanical properties are not well understood. In 
      particular, we know little about their relative speed and response to structural 
      obstacles in the template. Commercial retroviral RTs stop at many positions along 
      mixed sequence templates, resulting in truncated cDNA products that complicate 
      downstream analysis. By contrast, group II intron-encoded RTs appear to copy long 
      RNAs with high processivity and minimal stops. However, their speed, consistency 
      and pausing behavior have not been explored. Here, we analyze RT velocity as the 
      enzyme moves through heterogeneous sequences and structures that are embedded 
      within a long noncoding RNA transcript. We observe that heterogeneities in the 
      template are highly disruptive to primer extension by retroviral RTs. However, 
      sequence composition and template structure have negligible effects on behavior 
      of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in 
      a single pass, and displays synchronized primer extension at a constant speed of 
      25 nt/sec. In addition, it passes through stable RNA structural motifs without 
      perturbation of velocity. Taken together, the results demonstrate that 
      consistent, robust translocative behavior is a hallmark of group II 
      intron-encoded RTs, some of which operate at high velocity.
CI  - (c) The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic 
      Acids Research.
FAU - Guo, Li-Tao
AU  - Guo LT
AD  - Department of Molecular, Cellular, and Developmental Biology, Yale University, 
      New Haven, CT 06520, USA.
FAU - Olson, Sara
AU  - Olson S
AD  - Department of Genetics and Genome Sciences, Institute for Systems Genomics, UConn 
      Health, Farmington, CT 06030-6403, USA.
FAU - Patel, Shivali
AU  - Patel S
AD  - Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 
      CT 06520, USA.
FAU - Graveley, Brenton R
AU  - Graveley BR
AD  - Department of Genetics and Genome Sciences, Institute for Systems Genomics, UConn 
      Health, Farmington, CT 06030-6403, USA.
FAU - Pyle, Anna Marie
AU  - Pyle AM
AUID- ORCID: 0000-0001-9045-8872
AD  - Department of Molecular, Cellular, and Developmental Biology, Yale University, 
      New Haven, CT 06520, USA.
AD  - Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
AD  - Department of Chemistry, Yale University, New Haven, CT 06520, USA.
LA  - eng
GR  - R01 HG011868/HG/NHGRI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Nucleic Acids Res
JT  - Nucleic acids research
JID - 0411011
RN  - EC 2.7.7.49 (RNA-Directed DNA Polymerase)
SB  - IM
MH  - *Biotechnology
MH  - *RNA-Directed DNA Polymerase/genetics
MH  - *Sequence Analysis, RNA/methods
PMC - PMC9262592
EDAT- 2022/06/18 06:00
MHDA- 2022/12/15 06:00
PMCR- 2022/06/17
CRDT- 2022/06/17 10:11
PHST- 2022/06/01 00:00 [accepted]
PHST- 2022/05/26 00:00 [revised]
PHST- 2022/02/10 00:00 [received]
PHST- 2022/06/18 06:00 [pubmed]
PHST- 2022/12/15 06:00 [medline]
PHST- 2022/06/17 10:11 [entrez]
PHST- 2022/06/17 00:00 [pmc-release]
AID - 6609810 [pii]
AID - gkac518 [pii]
AID - 10.1093/nar/gkac518 [doi]
PST - ppublish
SO  - Nucleic Acids Res. 2022 Jul 8;50(12):6980-6989. doi: 10.1093/nar/gkac518.