Central Dogma Regulation of Gene Expression Genetic Exchange Microbial Genetics Central Dogma Regulation of Gene Expression Genetic Exchange

Precursors and “Building Blocks” of Nucleic Acids

Nucleotides polymerize to a nucleic acid by phosphodiester bonds. An organic ester: R-C-O-C-R A phosphodiester: R-C-O-P-O-C-R =O =O =O

Base Pair Complementation Pyrimidines: C & T (U in RNA) Purines: G &A G to C by 3 H-bonds A to T (U) by 2 H-bonds High G+C content of DNA means more H-bonds; requires more energy to separate (denature) duplex.

RNA Structure: Single Strand with Self Complementation hairpin loop structure (see transcription terminator region ) 5’-P 3’-OH A G C T T C A G C A T G

5’-P 3’-OH DNA Structure: Complementation of Two Antiparallel Strands Forms into a Double Helix Conformation 3’-OH 5’-P

DNA Replication It is semi-conservative; half from the “parent” half newly synthesized. It’s initiated at a replication fork; DNA must be unwound and unbound into two single strands. Parent strands become the templates for the new (complementary) strands.

DNA Polymerase Reaction It must add to an existing nucleic acid (RNA or DNA). Adds dNTP to the 3’-OH end of a new strand; it grows in the 5’→ 3’ direction. DNA-Poly III has “Proofreading” capability; can catch and fix mistakes most of the time by 3’→ 5’ exonuclease activity.

Enzyme Functions in Replication This is a simple cartoon; exaggerated to illustrate function. DNA Polymerase III Primosome (makes RNA primer) Ligase (connects O-fragments) DNA Polymerase I (also has RNase, 5’ → 3’ exonuclease, activity to remove RNA primer)

Replisome Two DNA Polymerase III stay together in a complex called the replisome. This requires the lagging strand to loop back onto its strands DNA Polymerase III. When the lagging strand DNA Polymerase reaches the previous Okazaki Fragment it drops off and then grabs the next RNA primer.

Prokaryote Chromosome Replication (OriC) Chromosome is of circular DNA, and supercoiled (extra twists in helix knots-up or condenses DNA when not replicating). Plasmids = extrachromosomal element; circular; 100-1000 times smaller than chromosome. Also supercoils when not replicating. Plasmid DNA replication like that of chromosome or by rolling circle replication (more later in discussing bacterial conjugation, “sex”). Termination (ter)

Replication Initiation Initiator proteins (IP; about 30) bind at repetitive sequences within the OriC site. DNA winds around IP-complex; induces separation of strand at adjacent AT rich site. Single strand binding proteins & helicases attach. Formation of two replication forks that open in opposite directions. ~30