Bacterial transcriptomics Flashcards
Whats the transcriptome
}Transcriptome is the complete set of RNA molecules produced in a cell (dynamic)
}”Everything changes and nothing remains still … and … you cannot step twice into the same stream“ Heraclitus
}Access to the transcriptome is dependent on genomic data
}Technologies: microarrays -> tiling arrays -> RNAseq
Affymetrix human genome chip
}Complete set of predicted genes from genome sequence data can be used to construct a microarray.
}Probing of chip with fluorescently labelled mRNA can be used to investigate expression levels of all genes simultaneously.
}40,000 gene fragments
tiling arrays
}A type of DNA microarray that uses a set of overlapping oligonucleotide probes that cover the whole genome at very high resolution.
}Requires hundreds of thousands of probes
}mRNA purification (see below) not critical
}Transcriptome maps higher resolution than traditional microarrays
} … lower resolution than RNAseq
General features of RNA seq
}NGS allows direct sequencing of transcriptomes to great depth
}Introduced 2008
}Key issue – relative mRNA/rRNA abundance (see next slide)
}mRNA to cDNA by reverse transcription
}Bacterial mRNA lacks poly A tail of euk mRNA used for cDNA production via oligo dT priming
}Alternative approaches
}Random priming hexamers
}artificially add poly A tail
}ligate RNA probe to act as priming site
}Generates millions of short (25 - 200bp) sequencing reads corresponding to RNA fragments of transcriptome
}Reads mapped to reference genome to identify expressed regions
}Potentially single base resolution
Classic operon theory
}Some transcription units contain two or more genes
}Genes co-transcribed by RNA polymerase yielding single RNA molecule
}Often gene products have related function
}Prokaryotic phenomenon
}Lac operon is classic example
Transcriptome discoveries : operon complexity
Intra-operon decaying expression (each expression of neighbour gene is in a lower intensity)
Approximately half of consecutive genes within operons in Mycoplasma pneumoniae have staircase expression pattern
Transcriptome discoveries – abundance of non-coding small RNAs
}Function in regulation of gene expression
}Can act in cis or trans
}Transcriptional or translational levels
}Can have multiple targets
}Example in quorum sensing lecture
}With RNAseq approaches many cis- and trans-acting examples of ncRNA being discovered in bacterial species
Transcriptome discoveries : abundant antisense
Abundance of antisense transcript inferred from bacterial RNA seq studies is astonishing
originally thought to be rare
Transcriptome discoveries : regulatory elements within mRNA
A lysine riboswitch is located upstream of a lysine transporter gene.
In the presence of lysine, transcription is prevented by a Rho-independent terminator.
Increasing numbers of riboswitches being identified with RNAseq approaches
Key discoveries of transcriptomics
}Transcriptome analysis has revealed considerable complexity
}Plethora of non-coding RNAs (sRNAs and antisense)
}Novel untranslated regulatory regions
}Complexity in operon structures
}Challenge will be to understand role of these newly discovered layers of regulatory complexity
