Transcription in bacteria is carried out by a multi-subunit RNA polymerase (RNAP) that is conserved from bacteria to humans. Promoter-specific transcription initiation depends on the ??subunit of RNAP, which must associate with the catalytically proficient core enzyme to form the holoenzyme. Historically defined as an initiation factor, ? can also remain associated with the transcription elongation complex and influence the transcription process during elongation. In particular, ? can induce early elongation pausing by establishing sequence-specific interactions with promoter-like DNA sequence elements within the initial transcribed region of a gene. The proposed research builds on the demonstration that such promoter-like pause- inducing elements can function to inhibit ? loss during the earliest stage of elongation, increasing the ? content of elongation complexes throughout the transcription unit. The results of bioinformatic analyses suggest that promoter-proximal ?-dependent pause elements are associated with at least 20% of all E. coli promoters, which implies that this mechanism for regulating the ? content of elongation complexes is likely a general feature of transcription in E. coli.
The first aim of the proposed research is to investigae the effects of ? retention on E. coli gene expression.
The second aim i s to investigate whether the potential roles for ? during transcription elongation extend beyond the primary ? factors and to determine whether different types of ? factors can participate in initiation and elongation on the same transcription unit (? "cross-regulation").
The third aim i s to investigate the mechanism by which promoter-proximal pause elements influence the ? content of downstream elongation complexes.
The fourth aim i s to investigate whether a specific ?-core interaction that may be essential for cell viability facilitates the release of ? from the DNA in the context of a paused early elongation complex. Together, the proposed experiments will lead to a deeper understanding of the roles of a classical initiation factor during transcription elongation, contributing to an accumulating body of evidence for the importance of post-initiation regulatory events in both prokaryotes and eukaryotes.

Public Health Relevance

RNA polymerase (RNAP), which is responsible for the process of transcription, is the central enzyme of gene expression. An important antimicrobial target, the bacterial RNAP has provided a crucial model for understanding transcription, and the principles that emerge from these investigations continue to inform the study of the many transcription-based processes that underlie human development and human disease. The proposed research is focused on an essential component of RNAP, the historically defined transcription initiation factor ?, which is emerging as a general transcription factor that influences all stages of the transcription cycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM044025-24
Application #
8641366
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
1990-04-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
24
Fiscal Year
2014
Total Cost
$626,078
Indirect Cost
$254,369
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Bikard, David; Jiang, Wenyan; Samai, Poulami et al. (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 41:7429-37
Montero-Diez, Cristina; Deighan, Padraig; Osmundson, Joseph et al. (2013) Phage-encoded inhibitor of Staphylococcus aureus transcription exerts context-dependent effects on promoter function in a modified Escherichia coli-based transcription system. J Bacteriol 195:3621-8
Deighan, Padraig; Pukhrambam, Chirangini; Nickels, Bryce E et al. (2011) Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex. Genes Dev 25:77-88
Twist, Kelly-Anne F; Campbell, Elizabeth A; Deighan, Padraig et al. (2011) Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the ?-subunit flap domain of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 108:19961-6
Hochschild, Ann; Lewis, Mitchell (2009) The bacteriophage lambda CI protein finds an asymmetric solution. Curr Opin Struct Biol 19:79-86
Stallings, Christina L; Stephanou, Nicolas C; Chu, Linda et al. (2009) CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence. Cell 138:146-59
Rao, Xiancai; Deighan, Padraig; Hua, Ziyu et al. (2009) A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the beta subunit and the primary sigma subunit. Genes Dev 23:1818-29
Yuan, Andy H; Nickels, Bryce E; Hochschild, Ann (2009) The bacteriophage T4 AsiA protein contacts the beta-flap domain of RNA polymerase. Proc Natl Acad Sci U S A 106:6597-602
Yuan, Andy H; Gregory, Brian D; Sharp, Josh S et al. (2008) Rsd family proteins make simultaneous interactions with regions 2 and 4 of the primary sigma factor. Mol Microbiol 70:1136-51
Deighan, Padraig; Diez, Cristina Montero; Leibman, Mark et al. (2008) The bacteriophage lambda Q antiterminator protein contacts the beta-flap domain of RNA polymerase. Proc Natl Acad Sci U S A 105:15305-10

Showing the most recent 10 out of 17 publications