Fundamentals: Nucleic acids, DNA replication, transcription, translation and application to molecular detection

Prokaryotic cell

Binary Fission
Bacteria reproduce asexually via binary fission Each daughter cell is an identical copy (or clone) of its parent cell

Microbial evolution 101

Generation 1 Generation 2 Generation 3 Generation N Ancestor Genotype Clones Clones Clones and Divergent Genotypes

Microbial genetics 101

What is DNA ? What types of DNA molecules are present in a bacterial cell? Whats the size of the genetic material for a typical bacterial pathogen ? How many genes does a bacterial pathogen have ? Whats the average size of a bacterial gene ?

What Is DNA?

Double Helix Structure and Antiparallel Orientation

Nucleotide = 5 carbon sugar + nitrogenous base + phosphate group

DNA is a polynucleotide

Promoter Ribosome binding site Open reading frame Start & Stop codons
Start codon ATG -35 -10

Constituents of a Gene

Stop codon TAG

Promoter Ribosome binding site

The Central Dogma

DNA
Molecular methods
DNA replication Transcription

mRNA
Translation

Classical methods

Protein/Enzymes
Post-translation

Toxins & other metabolites

DNA Replication
Topoisomerases remove superhelicity New DNA is synthesized in the 5 to 3 direction Replication begins at a the origin of replication (ori) Two replication forks proceed around the chromosome (bi-directional) until they encounter termination (ter) sites Replication is continuous on one strand and discontinuous on the other strand Chromosomes partitioned into two daughter cells during cell division

DNA Replication
Leading strand; continuous replication Lagging strand; discontinuous replication

Helicase; unwinds helix Single-stranded binding protein; binds single-stranded DNA prevent hybridization Primase; lays down RNA primers needed for DNA polymerase activity DNA polymerase I; remove RNA primers replace with DNA DNA polymerase II; DNA repair DNA polymerase III; major replication enzyme forms phosphodiester bonds Ligase; seals nicks by linking free 3 OH with 5 adjacent phosphate group Proofreading (DNA pol I & III) 3 to 5 exonuclease activity to remove incorrect base Incorrect base incorporated every 1x108 to 1x1011 bases

DNA Polymerase and PCR

DNA polymerase III Polymerase with or without 3 to 5 exonuclease proofreading activity
Taq (5 - 3 exonuclease activity); only degrades double stranded DNA while extending Vent (3 to 5 exonuclease activity; specialized Taq) Implications
Detection/subtyping methods Cloning

DNA Replication and Application to Molecular Detection

Polymerase chain reaction (PCR) or DNA photocopying
Simulate the natural DNA replication process to make copies of DNA in vitro Make many copies of specific DNA fragment(s) in vitro
Template, deoxynucleotidetriphosphates, primers, DNA polymerase, enzyme cofactors, and buffer

RNA v. DNA

RNA in the cell

Ribosomal RNA (rRNA) bulk of RNA in a cell 3 types (16s, 23s, and 5s) 3,000 copies in a cell Ribosomes; protein assembly during translation Messenger RNA (mRNA) 5-10% of RNA in a cell Almost as many types as there are genes Not stable in the cell; highly transcribed genes have a few hundred copies; half-life a few minutes (1 to 7 min.) Synthesized from DNA during transcription Move information contained in DNA to translation machinery Transfer RNA (tRNA) About 50 types Pick up amino acid & transport to ribosome during translation Small RNA (sRNAs) 50 - 200 nucleotides Regulatory roles (e.g., affect mRNA stability and translation)

The Central Dogma

DNA
Molecular methods
DNA replication Transcription

mRNA
Translation

Classical methods

Protein/Enzymes
Post-translation

Toxins & other metabolites

Transcription and translation are coupled in bacteria

Holoenzyme (RNA polymerase and sigma factor

5 3
tRNA Anticodon

Transcription

3 5
50S large subunit (23S and 5S RNA and proteins) 30S small subunit (16S RNA and proteins)

mRNA Ribosomes
Translation Direction

Translated Protein

A closer look at transcription

DNA used as template to synthesize complementary mRNA molecules RNA polymerase (pol) binds to promoter region in double-stranded DNA Sigma factors help RNA pol bind promoter & target genes to be transcribed -10 and -35 region 5 of transcription start site Local unwinding of double-stranded DNA RNA pol recognizes transcription start site RNA pol adds nucleotides 5 to 3 RNA pol termination RNA pol and RNA molecule released Rho-dependent Rho-independent (hairpin loop; termination sequence)

The Central Dogma

DNA
Molecular methods
DNA replication Transcription

mRNA
Translation

Classical methods

Protein/Enzymes
Post-translation

Toxins & other metabolites

A closer look at translation

Three ribosome sites: A site; entry of aminoacyl tRNA (except 1st aminoacyl tRNA or start codon, which enters at P site) P site; peptidyl tRNA is formed E site; exit for uncharged tRNA Shine-Dalgarno sequence or ribosome binding site recognized (5-10 bases upstream of start codon) Assembly of small and large ribosome subunits Amino acids added to carboxyl end of growing chain Protein exits ribosome through tunnel in large subunit Termination occurs when one of three termination codons moves into A site

Genetic code

Application to molecular detection in food microbiology

Molecular detection methods include assays that target nucleic acids (i.e., DNA and RNA) DNA detection methods

mRNA detection methods

Detect presence or absence of gene(s) or gene fragment(s) specific to the target organism Detection of universal gene or gene fragment (e.g., 16s rRNA) followed by DNA sequencing Detection of DNA does not differentiate between viable and nonviable organism mRNA is rapidly degraded and detection indicates presence of viable organism

PCR Applications
PCR detection particularly useful when
Classical detection too time-consuming Differentiation from closely related non-pathogenic organisms is difficult

Listeria monocytogenes
Only species in Listeria genera that is pathogenic to humans PCR assay targeted to detect hemolysin (hlyA) gene can detect presence and differentiate L. monocytogenes from other Listeria spp.

PCR Reaction Components

1 - Small quantity of DNA added to tube 2 - DNA polymerase 3 - Oligonucleotides (primers) 4 - Deoxynucleotidetriphosphate bases 5 - MgCl2 6 - Buffer 7 - Sterile ultrapure water