Timelyandaccurateregulationofgeneexpressionisrequiredforpropergrowth,development, andresponsetoenvironmentalstimuli.Acompleteunderstandingofthemechanisms employedtoregulategeneexpressionisnecessarytocombattheaberrantregulationthat underliesmanyhumandevelopmentalconditionsandailments,includingcancer.Thelong-term goalsofthisproposalaretodeterminethemechanicsofRNAsynthesisbyDNA-dependent multi-subunitRNApolymerases(RNAPs),theregulationimposedonRNAPbyconserved proteinfactors,selecttemplatesequencesandchromatin-barriers,andtospecifically characterizetheavailablemechanismstohaltRNAsynthesisandterminatetranscription.The proposedexperimentstakeadvantageofanestablishedinvivoandinvitroarchaeal transcriptionsystemfromthemarinehyperthermophilicorganismThermococcuskodakarensis. Archaeaoffertheadvantagesoffarlesscomplexitybuthomologyinmanyfeaturesofhuman molecularbiology,specificallyconservationofRNAPstructureandfunctionandhistone-based chromatin.Wewillcharacterizefactor-dependentandintrinsictranscriptiontermination, polarity,theconsequencesofDNAdamageontranscriptionelongationandtermination,andthe roleofglobaltranscriptionregulationinarchaealphysiology.Theresultsobtainedwill determinethestructuresandsequencesthatsupporttranscriptionelongationcomplexstability, describethemechanicsofterminationandallowcomparisonsofterminationmechanismsin eachdomainoflife,andprovideinsighttocontrolandultimatelymanipulategeneexpressionto counteravarietyofdiseasesandailmentsaffectinghumanhealth.

Public Health Relevance

Acompleteunderstandingofthemechanismsemployedtoregulategeneexpressionis necessarytocombattheaberrantregulationthatunderliesmanyhumandevelopmental conditionsandailments,includingcancer.Thelong-termgoalsofthisproposalareto determinethemechanicsofRNAsynthesisbyDNA-dependentmulti-subunitRNApolymerases (RNAPs)andtospecificallycharacterizetheavailablemechanismstohaltRNAsynthesisand terminatetranscription.Theresultsobtainedwilldeterminethestructuresandsequencesthat supporttranscriptionelongationcomplexstability,describethemechanismsofterminationand allowcomparisonsofterminationmechanismsineachdomain.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM100329-07
Application #
9444953
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
2012-02-01
Project End
2022-04-30
Budget Start
2018-07-01
Budget End
2019-04-30
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Speed, Michael Clayton; Burkhart, Brett W; Picking, Jonathan W et al. (2018) An Archaeal Fluoride-Responsive Riboswitch Provides an Inducible Expression System for Hyperthermophiles. Appl Environ Microbiol 84:
Gehring, Alexandra M; Santangelo, Thomas J (2017) Archaeal RNA polymerase arrests transcription at DNA lesions. Transcription 8:288-296
Wang, Peng; Yu, Zhuoteng; Santangelo, Thomas J et al. (2017) BosR Is A Novel Fur Family Member Responsive to Copper and Regulating Copper Homeostasis in Borrelia burgdorferi. J Bacteriol 199:
Gehring, Alexandra M; Astling, David P; Matsumi, Rie et al. (2017) Genome Replication in Thermococcus kodakarensis Independent of Cdc6 and an Origin of Replication. Front Microbiol 8:2084
Mattiroli, Francesca; Bhattacharyya, Sudipta; Dyer, Pamela N et al. (2017) Structure of histone-based chromatin in Archaea. Science 357:609-612
Burkhart, Brett W; Cubonova, Lubomira; Heider, Margaret R et al. (2017) The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis. J Bacteriol 199:
Gehring, Alexandra M; Sanders, Travis J; Santangelo, Thomas J (2017) Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis. Bio Protoc 7:
Walker, Julie E; Luyties, Olivia; Santangelo, Thomas J (2017) Factor-dependent archaeal transcription termination. Proc Natl Acad Sci U S A 114:E6767-E6773
Gehring, Alexandra M; Walker, Julie E; Santangelo, Thomas J (2016) Transcription Regulation in Archaea. J Bacteriol 198:1906-1917
Gehring, Alexandra M; Santangelo, Thomas J (2015) Manipulating archaeal systems to permit analyses of transcription elongation-termination decisions in vitro. Methods Mol Biol 1276:263-79

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