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AQ curious about

Promoters?

Joshua Baker, Haley Everroad, and Madison Powell

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Introduction

  • Promoters are an important part of mRNA transcription and indicate the beginning of an operon
  • Potential promoters are often found in gaps between a reverse gene and a forward gene
  • Located before the gene’s DNA sequence, the promoter is a region of DNA that allows RNA polymerase to bind and begin the process of transcription

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The Main Question

  • Many phages in the AQ cluster have very similar nucleotide sequences and have a gap between genes 10 & 11 and genes 83 & 84 where potential promoters could be located
  • What promoters exist in the AQ cluster? Specifically identify a consensus sequence between genes 10 & 11 and genes 83 & 84
  • Our group focused on AQ phages Amigo, Anansi, Gorgeous, Rings, and SorJuana

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DY Wu et al. Nature 462, 1056-1060 (2009) doi:10.1038/nature08656

Actinobacteria

Gammaproteobacteria

Take away:

E. coli and Arthrobacter are not very related. Are the promoter sequences conserved?

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MEthods

  • Examine nucleotide sequences between genes 10 & 11 and genes 83 & 84 of annotated AQ phages
  • Run a sigma 70 promoter predictor in DNA Master
    • Sigma 70 employs the E. coli consensus sequence TTGACA_17_TATAAT
  • Compile data
  • Develop a consensus sequence using averages
  • Verify the presence of promoters with a termination prediction program
  • Compare to AN consensus sequence and E. coli consensus sequence

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Results

Average Gap Size Between Genes

10 & 11: 17.64285714

Average Gap Size Between Genes

83 & 84:

17.48484848

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2

3

4

5

6

Gene 10/11 -10 Promoter

C/A

A

T

A

T/A

T

Gene 83/84 -10 Promoter

T

A

T

A

A/T

T

-10 Promoter

Consensus

T

A

T

A

A/T

T

Gene 10/11 -35 Promoter

T/G

T

G

A

A/G

A

Gene 83/84 -35 Promoter

T

T

G

A/C

C/T/A

A

-35 Promoter Consensus

T

T

G

A

A/C

A

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Results

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2

3

4

5

6

-10 CONSENSUS

T

A

T

A

A/T

T

-35 CONSENSUS

T

T

G

A

A/C

A

1

2

3

4

5

6

-10 CONSENSUS

T

T

G

A

T

T

-35 CONSENSUS

T

T

G

A

C

G

1

2

3

4

5

6

-10 CONSENSUS

T

A

T

A

A

T

-35 CONSENSUS

T

T

G

A

C

A

AQ PHAGES

AVERAGE GAP:

17.56385281

AN PHAGES

AVERAGE GAP:

16.82692308

E. coli

AVERAGE GAP:

17

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Rho - Independent Transcription Terminase Prediction

  • Verified the presence of promoters by predicting the terminating region of entire operons
  • Used ARNold predicting software
    • Functions by using two complementary programs, Erpin and RNAmotif.
  • Erpin is given a structure-annotated alignment of 1200 terminator sequences from B. subtilis and E. coli
  • RNAmotif recognizes E. coli terminators, however it can be applied to find terminators from any species
    • To provide a uniform scoring scheme for Erpin and RNAmotif hits, ARNold computes the free energy of the predicted terminator stem-loop structure using RNAfold. This free energy value may be used as a confidence value for predicted terminators.

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Gene 10 & 11 Promoter Verification

Predicted Promoter Region

Reverse Strand Predicted Terminase (1364 bp)

Score: -16.10

Predicted Protein Coding Sequence

Forward Strand Predicted Terminase (32151 bp) Score: -16.40

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Gene 83 & 84 Promoter Verification

Predicted Promoter Region

Predicted Protein Coding Sequence

Reverse Strand Predicted Terminase (47445 bp)

Score: -17.60

Forward Strand Predicted Terminase (32151 bp) Score: -16.40

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10/11 Reverse Strand Termination Sequence

10/11 Forward Strand Termination Sequence

83/84 Reverse Strand Termination Sequence

83/84 Forward Strand Termination Sequence

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Discussion

  • When comparing our consensus sequence to that of E. coli, the sequences are nearly identical
  • Comparison of our consensus sequence to that of the AN phages reveals less similarities
  • Similar consensus sequences could indicate similar promoters in bacteria and phages
  • Small sample size could have affected results
  • Presence of a terminase verifies that a promoter exists in the areas studied

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Conclusion

  • Due to the supporting evidence that was gathered, it can be argued that a promoter exists between genes 10 & 11 and genes 83 & 84 of phages in the AQ cluster
  • Identification of promoters in phages leads to a better understanding of prokaryotic transcription and subsequent gene expression
  • More insight into phages could lead to important advances in the medical field, such as the development of phage therapy

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REFERENCES

  • Harley, C B, and R P Reynolds. “Analysis of E. Coli Promoter Sequences.” Nucleic Acids Research 15.5 (1987): 2343–2361. Print.
  • Adhya, S et al. “Location, Function, and Nucleotide Sequence of a Promoter for Bacteriophage T3 RNA Polymerase.” Proceedings of the National Academy of Sciences of the United States of America 78.1 (1981): 147–151. Print.
  • Shlomit Lisser and Hanah Margalit. “Compilation of E. coli mRNA promoter sequences.” Nucleic Acids Research 21.7 (1993): 1507-1516. Print.

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ACKNOWLEDGEMENTS

  • We would like to thank Tamarah Adair Ph.D. for granting us this opportunity, as well as Lathan Lucas, Jennifer Wilson, and Ashley Young for their assistance in lab. Furthermore we would like to thank the SEA-PHAGE program for their funding.

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Questions?