Central dogma: Information flow in cells

Nucleotides

• Pyrimidine bases: Cytosine (C), Thymine (T), Uracil (U, in RNA)

• Purine bases: Adenine (A), Guanine (U)

Prokaryotic gene coding

Eukaryotic processing of rRNA

A-T hydrogen bonding

G-C hydrogen bonding

Genetic Elements

• Prokaryotes: Chromosome, plasmid, viral genome, transposable elements

• Eukaryotes: Chromosomes, plasmid, mitochondrion or chloroplast genome, viral genome, transposable elements

Melting of DNA

• Melting means separation of two strands from the heteroduplex

• Melting temperature of DNA is dependent on the relative number of AT and GC pairs

• Melted DNA can hybridize at temperatures below melting temperature– This process can be used to test relatedness between

species (interspecies DNA-DNA hybridization)– It is also possible to reanneal DNA with rRNA to test

relatedness of one species rRNA with the rRNA genes of another species

**DNA structure overview**

• complementary strands (antiparallel)

• 3 Angstrom separation of hydrogen bonds

• sugar phosphate backbone held together with hydrogen bonding between bases

• size is expressed in nucleotide bases pairs. E. coli has 4600 kbp. (E. coli chromosome is > 1mm, about 500X longer than the cell itself. How can the organism pack so much DNA into its cell?

• each bp takes up to 0.34nm, and each helix turn is 10bp(or 34 Angstroms), therefore how long is l kb of DNA? and how many turns does it have?

• supercoiled DNA (DNA-binding proteins)

DNA Organization

• In prokaryotes: naked circular DNA with negative supercoiling– Negative supercoiling is introduced by DNA gyrase

(topoisomerase II)– Topoisomerase I relaxes supercoiling by way of single-

strand nicks

• In eukaryotes: linear DNA packaged around histones in units called nucleosomes– The coiling around histones causes negative

supercoiling

Restriction and modification

DNA Replication: addition of a nucleotide

Semiconservative replication

Initiation of DNA replication

Origin of replication= oriC = ~300bpTemplates, primers, polymerase, primase

DNA Replication

Bidirectional replication

Okazaki fragments

Proofreading by DNA polymerase III

Replication overview

• 1. origin of replication+ 300 bases, recognized by specific initiation proteins = replication fork

• 2. bidirectional, therefore leading and lagging strands

• helicase unwinds the DNA a little (ATP-dependant) • single-strand binding protein prevents single strand from reannealing • Primase, DNA polymerase III and DNA polymerase I (also 5' to 3'

exonuclease activity), ligase • Okazaki fragments • Topoisomerases, and supercoiling regulation

• 3. Proofreading (3 to 5' exonuclease activity by DNA pol III)

DNA Sequencing

Transcription

• RNA plays an important role

• tRNA, mRNA, rRNA

• Name three differences between chemistry of RNA and DNA

• RNA has both functional and genetic roles

Initiation of TranscriptionPribnow box=tataat

Transcription

Completion of transcription

Example of termination sequence

More transcription

• Polycistronic mRNA

• How can mRNA be used in microbial ecology?

• Antibiotics and RNA polymerases

RNA processing

• Removal of introns

• Ribozymes (nobel prize-Tom Cech and Sid Altman)

• RNA-splicing enzymes

• Origins of life? Which came first RNA or DNA?

The genetic code

• Notice that the wobble base generally makes minor changes in the amino acid

• AUG is the start code (formyl methionine) for bacteria

• UAA, UAG, UGA are stop codons

• Specific tRNA for each other codon

tRNA associated with codon

~60 specific tRNAsin prokaryotes

mRNA, tRNA and ribosomesShine Dalgarno sequenceGTP and Elongation Factors (EF)

Growing protein polymer

Translocation

Role of rRNA in protein synthesis

• Structural and functional role• 16S rRNA involved in initiation

– Base pairing occurs between ribosome binding sequence on the mRNA and a complementary seq on the 16S rRNA

• 23S rRNA involved in elongation– Interacts with EFs

Overview of today

• Summarized basic DNA structure

• DNA replication

• DNA sequencing

• Transcription

• RNA processing

• Translation

• Role of rRNA in protein synthesis