Clustered Repeats and Regulatory Sites - PowerPoint Presentation

Slide1

Clustered Repeats and Regulatory Sites

Abdulrahman

Alazemi

,

Shahroze

Abbas, Liam Lewis, Donald Ta, Ann VoSlide2

Overview

Clustered repeats

Wide variety of functions

History largely unknown

Regulatory Sites

A segment capable of altering expression of specific genes

Various classifications of regulatory sequences

Found in non-coding regions

Functions at the transcriptional levelSlide3

Identification

Consensus sequences

Utilize PSSM

How?

Determine consensusSlide4

Clustered repeats and potential regulatory sequences

Abdulrahman

AlazemiSlide5

Background;

Transcription factors?

Activators Vs Repressors.Slide6

Thoughts;

- My questions;

Can I apply a known method/tool with a known results to other phage and get the same/similar result?Slide7

The known case;

Examine the proven Repressor and Cro binding sites (operators) of Phage Lambda.

Bioinformatics method in the notes.

First “Name of Lambda in BioBike”.

Go to BioBike/Phantome.Slide8

The known case;

Second; Motifs in for the upstream sequence of phage Lambda.

Labeled, DNA, Multiple-Hits-ok.Slide9

The known case;

Results of motifs in.

More than one interesting case.

Motifs 1, 2 , 3.Slide10

The known case;

BioBike function;

Description-analysis

submenu, Genes-

proteins menu.

Now, we have an

idea about where

to look.Slide11

The known case;

Used the function sequence-of from Genome menu.

Go to the specific region in the genomeSlide12

The known case;

Finding the operators.

Directly Vs inversion of.Slide13

Phage Lambda map;Slide14

Thoughts;

- My concern/focus;

Would I find some sort of generality between operators of different phages?Slide15

My experiment;

Twenty one random phages of different phage families.

Eight of them don't have repressors. (eliminated)

Three of the 13 phages left didn't display a map because of linear amplicon.

Ten phages out of 21 went through all the steps of the method/tool successfully and gave me back out come that I can work with.

Three out of 10 have similar results to phage Lambda. Slide16

Outcome analysis;

- Similar to phage lambda:

-Bacillus-phage-1Slide17

Phage

Bacillus-phage-1 map;

Slide18

Outcome analysis;

-Similar to phage Lambda:

-Listeria-phage-A006Slide19

Phage Listeria-phage-A006 map;Slide20

Outcome analysis;

-Similar to phage lambda:

-

Lactobacillus-johnsonii-prophage-Lj928Slide21

Phage

Lactobacillus-johnsonii-prophage-Lj928 map;

Slide22

First conclusion;

- Out of 21 or 10 phages, only 3 phages are similar to phage Lambda.

- Less than 50%.

- No Generality.

- Appropriate conclusion;

- Phage Lambda, Bacillus-phage-1, Listeria-phage-A006 , and

Lactobacillus-johnsonii-prophage-Lj928 have a similarity/generality between their operators that the repressors bind to.

Slide23

Inspiration;

- Dead end.

- The articles !!!!

- Extend my research.

- look for something interesting.Slide24

First interesting case;

- In Burkholderia-phage-Bcep1.

- Six similar sequence in one intergenic region

- another 6 similar sequences in another intergenic region.

- Palindromic sequences.

- 6 or 3 sequences ?

- Bacillus-phage-1 is similar to Burkholderia-phage-Bcep1 somehow.

Slide25

First interesting case;Slide26

Phage Burkholderia-phage-Bcep1 map;Slide27

Second interesting case;

- In phage Clostridium-phage-39-O.

- Eight nucleotides sequence (

TTACTACA)

repeated 10 times in one intergenic sequence.

- Again the same sequence repeated 8 times in another intergenic sequence in another place on the phage.Slide28

Second interesting case;Slide29

Phage Clostridium-phage-39-O map;Slide30

Conclusion;

- Goals;

- Pick one interesting case.

- Research it.

- Make sense of it.Slide31

A. Comparison of Pseudomonas

putida

and

Azotobacter

REP sequences

Donald TaSlide32

REPs

Repetitive

Extragenic

Palindromic Sequences

Found mainly in abundance in

Enterobacteriaecae

Can be anywhere around 20 to 40

nt

long

Clustered into structures called BIMEs (bacterial interspersed mosaic element) as two inverted tandem repeats separated by a short linker of variable lengthSlide33

What do REPs do?

Regulate Gene Expression

Structuring DNA

Specific target sites for bacterial insertion sequences

Possibly more that are undiscoveredSlide34

Previous Study

I.

Aranda-Olmedo

2002 used BLAST (Basic local alignment search tool) to find regions of local similarity between sequences downloaded from the National Center for Biotechnology Information (NCBI)

Used database with all

contigs

of Pseudomonas

putida

already available in The Institute for Genome Research

Developed their own program to screen all of the strains against the 35

nt

sequence 5’-CCGGCCTCTTCGCGGGTAAGCCCGCTCCTACAGGG-3’Slide35

Results of that StudySlide36

Implications from that study

They suggest that the 35

bp

element they found is species specific in P.

putida

First time that REP sequences have been described and characterized in a group of non-

enterobacteriaceaeSlide37

What am I doing?

Comparing REP sequence element of Pseudomonas

putida

KT2440 with

Azotobacter

vanlandii

Why?

Order

Pseudomonadales

Used the REP element that is most common among Pseudomonas species “

GCGGGnnnnCCCGC

”Slide38

Methods

Used built-in functions of

BioBike

to scan a sequence for possibly loose matches of a pattern

“****GCGGG****CCCGC****” sequence iterated over the sequence of the organism of interest and then whenever there was a match it was displayed on the output

“*” means an unspecified amino acidSlide39

Findings

52 sequence hits in

Azotobacter

vanlandii

that appear to have the same conserved region found in Pseudomonas

putida

The species share similar REP elements with the same conserved central palindromic region

“GCGGG****CCCGC”Slide40

OutputSlide41

Significance

REP sequences mainly found abundantly in

Enterobacteriacaea

Study by

Bao

Ton-Hoang 2012 suggested that

transposases

could’ve been responsible for the proliferation of REP sequences in the genomes of bacteria in

Enterobacteriacaea

Possibly suggest a similar origin of REP sequences/elements for Pseudomonas and

Enterobacteriacaea

?Slide42

Problems?

Found 2 hits in E. Coli K-12 that had the REP element

Maybe suggests similar origin?

Could be just a fluke/just by chance that these two organisms share the same REP element in abundance

Past Study found 804 REP sequences with that REP element in Pseudomonas

putida

I found 52 in

Azotobacter

vanlandiiSlide43

Possible plans of the future/near future?

Compare with other bacteria in the order of

Pseudomondas

to see if I get similar results

Possibly try to find a link to how REP sequences started proliferating in bacteria outside of

EnterobacteriacaeSlide44

Positional Preference of Rho-Independent Transcriptional Terminators in E. Coli

Ann VoSlide45

Transcriptional Terminators

Rho-independent

Specific activities poorly

understood

Occurs

in ssDNA and

RNA

Unique characteristics:

T

-Tract: 12-15

nt

GC-rich stem: 4-18

ntSlide46

Transcriptional Terminators

Available algorithms:

RNAMotif

TransTermHP

ARNold

About 317 natural terminators found in E.

Coli

Lai et al. (2013) found a positional preference between other regulatory sequences

Do transcriptional terminators have a positional preference relative to the end of the gene?Slide47

ARNold

Erpin

Scores input sequences

Compares against 1,200 known terminators from

Bacillus

subtilitis

and

Escherichia coli

RNAMotif

Used descriptors to find possible terminators

Scores free energy of hairpin formationSlide48

Matching Sequences

BioBIKE

/

PhAnToMe

Extracted the 50 nucleotides following every gene

Python

Compared sequences to terminators

Calculated distance to terminator

ARNold

3248 possible terminators

BioBIKE

5341 downstream sequences

Python

126 terminators

CAGGACGGTTTACCGGGGAGCCATAAACGGCTCCCTTTTCATTGTTATCA ACGGTTTACCGGGGAGCCATAAACGGCTCCCTTTTCATTGTTAdownstream sequenceterminatorSlide49
Slide50
Slide51
Slide52

Conclusion

Appear to exhibit some degree of positional preference

Reasons remain unclear

Further studies:

Length of terminator

Function of operonsSlide53

References

Chen, Ying-

Ja

et al. “Characterization of 582 Natural and Synthetic Terminators and Quantification of Their Design Constraints.”

Nature methods

10.7 (2013): 659–64. Web. 20 Mar. 2014.

Ermolaeva

, M D et al. “Prediction of Transcription Terminators in Bacterial Genomes.”

Journal of molecular biology

301.1 (2000): 27–33. Web. 4 Apr. 2014.

Kingsford, Carleton L,

Kunmi

Ayanbule

, and Steven L Salzberg. “Rapid, Accurate, Computational Discovery of Rho-Independent Transcription Terminators Illuminates Their Relationship to DNA Uptake.” Genome biology 8.2 (2007): R22. Web. 17 Apr. 2014.Lai, Fu-Jou et al. “Identifying Functional Transcription Factor Binding Sites in Yeast by Considering Their Positional Preference in the Promoters.” PloS one 8.12 (2013): e83791. Web. 10 Apr. 2014.Lau, Lester F et al. “A Potential Stem-Oop Structure and the Sequence CAAUCAA in the Transcript Are Insufficient to Signal Q-Dependent Transcription Termination at XtR1.” 12.2 (1984): 1287–1299. Print.Macke, T J et al. “RNAMotif, an RNA Secondary Structure Definition and Search Algorithm.” Nucleic Acids Research 29.22 (2001): 4724–35.Mooney, Rachel Anne, and Robert Landick. “Building a Better Stop Sign: Understanding the Signals That Terminate Transcription.” Nature Methods 10.7 (2013): 618–619. Web. 21 Mar. 2014.Naville, Magali et al. “ARNold: A Web Tool for the Prediction of Rho-Independent Transcription Terminators.” RNA Biology 8.1 (2011): 11–13. Web. 8 Apr. 2014.Slide54

Resemblances and differences between promoter sequences in

E. coli

and

S.

enterica

Liam LewisSlide55

Inspiration

Novel sequence-based method for identifying transcription factor binding sites in prokaryotic genomes

Results found promoters with high probability Slide56

Background of Promoter sequences

Regulatory Elements

-35 and -10 consensus sequence

Sigma factor + RNA Polymerase Slide57

Program used to identify promoters

PePPER

Uses PSSMs and Hidden

markov

Models

Algorithm is universal for prokaryotesSlide58

Biobike implementation

Biobike

to compare both outputs from

PePPER

.Slide59

What’s next?

Comparison of results

Biobike

algorithm to accurately predict promotersSlide60

Comparison of Repressor-Operator Sequences in Lambda and other Temperate Phages

Shahroze AbbasSlide61

Repressor Sequences in Lambda

Two possible life cycles, dependent upon either Lambda repressor or

Cro

repressor.

cI

repressor maintains lysogenic state

Cro

repressor initiates a switch to the lytic state

Significance of

intergenic

sequences and neighboring genes to determine ‘hypothetical proteins’ in other organisms similarSlide62

Comparison of Repressor Sequence

Lambda repressor sequence tested for occurrence in other phages

Enterobacteria

phage

Sfl

Enterobacteria

phage HK244

Enterobacteria

phage HK542

Enterobacteria

phage HK544

Enterobacteria

phage HK 106

Enterobacteria

phage CL707Slide63

Still to come…

Analysis of operator sequences

Comparison of

cro

repressor in other phages

Trend or pattern to determine function of neighboring proteins in other phages

Trend or pattern in

sequences between phages