Comparative genomics analysis of NtcA regulons in cyanobacteria: Regulation of nitrogen assimilation and its coupling to photosynthesis

Wen-Ting HuangJau-Chi Huang

Zhengchang Su, Vitctor Olman, Fenglou Mao and Ying Xu

Outline

• Introduction• Method• Result• Conclusion

Introduction

• DNA transcription

• mRNA

5’

5’

3’

3’

introncoding region

coding region

5’

5’

3’

3’

5’ 3’

upstream

downstreamtranscription direction

Regulatory region RNA polymerase binding site

Regulation elementsRNA polymerase

• cis-regulation– Regulatory regions of genes

and the regulated genes are on the same chromosome

• Phylogenetic footprinting – Identifies regulatory elements

by finding regions in a set of orthologous non-coding DNA sequences from multiple species.

• Cyanobacteria – bacteria – live in the water– Gram-Negative, oxygenic

phototrophs– Nitrogen control in

cyanobacteria is mediated by NtcA

http://www.ucmp.berkeley.edu/bacteria/cyanointro.html

• NtcA– A protein which regulates the

assimilation of nitrogen.

• NtcA binding site– Base Motif “GTAN8TAC”– ~14 bps– Intron– Nitrogen fixation related genes– -31 downstream has -10 σ70like

box “TAN3T”

High false positive rate

• Too short to identify• 3 methods:

– Coding region– -10 like box– Othologous genes

Materials

• Nine sequenced cyanobacteria genomes were downloaded from the GenBank.

• ftp.ncbi.nih.gov/genomes/Bacteria/

Method

• Step 1:– Prepare training sets– Get the profiles(GTAN8TAC,

TAN3T)

• Step 2:– Scan genomic sequences and score

each motif.

• Step 3:– Decide the cutoff.

Known

• Possible NtcA binding sites (GTAN8TAC)

– Appear in the upstream intergenic regions

– In many cases, there is a –10 like box (TAN3T) in the 31bp downstream regions of the NtcA binding site

transcription unitupstream

31bptranscription unit

Prepare training sets

• They chose 11 genes which are known to be regulated by NtcA from the nine cyanobacterial genomes.

• They used phylogenetic footprinting and identified 51 putative NtcA binding sites.

• These 51 sites constitute the training set A1 for the NtcA binding site.

• The –31 bp downstream regions are further searched for a –10 like box and form the training set B1

A1 & B1

A2&B2

• They collected 12 experimentally verified NtcA binding sites and their downstream from seven other cyanobacteria.

• They also included the sites that failed to find by phylogenetic footprinting.

Profiles

• They combined A1 and A2 to construct the profile of NctA binding sites.

Profiles

• They combined B1 and B2 to construct the profile of –10 like boxes.

Scan genomic sequences

upstream

transcription unit transcription unit

GTAAAGTTAAGTTCCTTCAAAGCATTCGTGG

TTAAAGTTAAGTTCTTTTAAAGCTTTCGTGG

l

ii

thM ihq

ihipItS

11 )]([

)](,[lnmax)(

Scan genomic sequences

upstream

transcription unit transcription unit

GTAAAGTTAAGTTCCTTCAAAGCATTCGTGG

TTAAAGTTAAGTTCTTTTAAAGCTTTCGTGG

l

ii

thM ihq

ihipItS

12 )]([

)](,[lnmax)(

The scoring functions

z

jMjMzM StS

1....1 )(

Orthologous genes

• The presence of similar motifs in the regulatory regions of the orthologous genes can increase the prediction accuracy.

• They predicted two genes in two genomes to be orthologous to each other if they are a pair of reciprocal best hit in BLASTP searches.

Orthologous genes

upstream

transcription unit

Cutoff

• The largest score for the genome to include all the binding sites from that genome in the training sets.

• P-value– p[S(CU)>sc]<0.01 or 0.05

Analysis

Analysis

“GTA________TAC” “TA___T”

Niche of NtcA in cyanobacteria … ?• Some genes bear NtcA

promoters might coordinate photosynthesis and nitrogen fixation.

• RNA polymerase σ-factor in cyanobacteria might bear an NtcA promoter and regulated by NtcA.

Conclusion

• The false positive rate is reduced from 8.2 to 90.9 fold.

• Some binding sites might be missed due to the lack of orthologues in the other genomes.

• NtcA promoters are found for many genes involved in the various stages of photosynthesis process.

Thank You