L21: Bacterial Genomes Overview Flashcards
Genes
Working subunits of DNA containing particular set of instructions -> encode gene products (tRNA, rRNA, mRNA/polypeptide)
Represented as arrows
Found on both strands of DNA
Generally do not overlap
Entire nucleic acid sequence necessary for expression of gene product
Promoter
-35 box to -10 box
Region where RNA pol binds to initiate transcription
Shine Dalgarno sequence
Region where ribosomes bind to initiate translation of protein
5’ untranslated region
Between transcription start +1 to start translation codon ATG
3’ untranslated region
Between stop codon (TAA, TGA, TAG) and transcription termination signal
Gene organisation in bacteria
Polycistronic mRNA contains multiple open reading frames that will be replaced separately to produce separate proteins
In bacteria, mRNA not spliced -> one open reading frame = one protein (polypeptide)
Some proteins can be rearranged through splicing after translation: proteins that can self-splice have inteins (intervening sequences) flanked by exteins
Inteins are removed and exteins are joined together
Automated Sanger DNA sequencing using chain termination method
- Microorganisms isolated in pure culture in lab
- Genomic DNA extracted and sheared into fragments
- Reaction incubated at 95 degrees-> template becomes ss. Reaction is cooled to 65degrees and primer will bind to template sequence where it matches 100%. Reaction incubated at 72 degrees and Taq pol extends primer using template as guide -> series of short ss DNA products that are terminated with di-deoxynucleotide
- Reaction mixtures separated by capillary electrophoresis based on size. Capillary electrophoresis can separate fragments by 1bp
- ddNTPs labelled with fluorophore -> pass through laser placed at end of gel. Excitation of fluorophore detected by detector instrument and converted into histogram (sequence output). Since every fragment is different by 1bp and fluorophore indicates which nucleotide was at then end -> shows actual sequence of template DNA
Create genome
At end of capillary electrophoresis step -> have 100,000s of short read fragments of 150 or so of bp
Info is fed into bioinformatics pipeline that:
- Removes primer sequence
- Fragment alignment: compiles fragments on overlapping similarity -> longer contigs of up to 20 kb
- Gap closure and editing: contigs aligned into scaffold assembling. Gaps can be filled in -> complete genome
Visualising genomic data. Escherichia coli O157:H7
Worldwide threat to public health
Cause many outbreaks of haemorrhagic colitis, including fatalities from haemolytic uraemic syndrome
Strain EDL933 genome sequenced to: identify candidate virulence genes, develop better methods of strain detection, advance understanding of E.coli evolution by comparison of EDL933 with lab strain E.coli K12 (MG1655)
Pan (or supra-) genome
Global gene repertoire of bacterial species that comprises sum of core and dispensable genome
Core genome: pool of genes shared by all strains of same bacterial species
Dispensable genome: genes shared by some but not all strains
Unique genome: only found in that one strain
Phylogenetic tree
Ribosomal gene encoding 16 S component is highly conserved
Function of 16S: universal -> changes in DNA sequence very rare over evolutionary time scales
Mapping changes of sequence of 16S rDNA: phylogenetic tree
Each branch indicates change in sequence. Longer branch, more changes
Analysis of complete metagenomes steps
- DNA isolation from microbial niches
- Construction of DNA libraries: ‘small-insert’ plasmid libraries; ‘large-insert’ cosmid, BACS libraries OR more commonly DNA pyrosequencing, Illumina sequencing and other technologies which do not require cloning (avoids bias that may be introduced by cloning efficiency variations)
- Mining for DNA sequences of interest: sequence based analyses (taxonomy), functional analyses (metabolic activities of microbial community can be explored), novel biotechnological applications
Example of metagenomic studies: the Human microbiome
Aim: sequence entire human microbiome of 250 people (nasal passages, ora cavities, skin, GT and urogenital tract)
Compare this to environment
Each environment has fingerprint of phyla at different abundances
Of 52 known phyla only 5-7 represented in human gut microbiome- environmental niches are highly selective
Transcription and translation
DNA sequences written in 5’ to 3’ direction
‘Coding’ or ‘sense’ DNA strand is written in 5’-3’ direction and is same as mRNA
DNA template strand is read by RNA pol in 3’ to 5’ direction from promoter to terminator
Transcribed mRNA is written in 5’ to 3’ direction
Translation of mRNA into protein begins with ribosome recognising ribosome binding site
tRNAs donate AA to growing peptide by recognise 3 base codon
Steps of transcription
Initiation: RNA pol binds promoter and initiates transcription at start point
Elongation: successive addition of ribonucleotides to RNA strand
Termination: completed mRNA transcript released
