Bacterial Genome Replication

What is a chromosome, a haploid, and a diploid?

• DNA package made of DNA and proteins

• haploid - one set of chromosomes

• diploid - 2 sets of chromosomes, one from each parent, formed through mitosis

Define the types of RNA (mRNa, rRNA, and tRNA)

• mRNA - messenger RNA, carries the genetic instructions from DNA to the ribosome, where proteins are made.

• rRNA - ribosomal RNA, combines with proteins to form ribosomes, helps read mRNA and link amino acids together during translation

• tRNA - transfer RNA, transfers amino acid to protein chain during translation by matching to mRNA codon

What is the central dogma?

genetic information flows from DNA to RNA to protein, through transcription and translation

What is replication?

DNA copied

What is transcription and translation?

• transcription - genetic information contained within DNA is re-written into messenger RNA

• translation - genetic code contained within a mRNA molecule is decoded to produce a specific sequence of amino acids in a polypeptide chain

What are the 4 levels of protein structure?

• primary structure – the sequence of amino acids in a polypeptide chain.

• Secondary structure – local folding into structures, held by hydrogen bonds.

• Tertiary structure – the overall 3D shape of a single polypeptide, formed by interactions between R-groups (like ionic bonds, disulfide bridges, hydrophobic interactions).

• Quaternary structure – the structure formed when multiple polypeptide chains (subunits) come together to form a functional protein.

what is a plasmid?

extracellular DNA not vital for existence, can be transferred to other organisms

Describe the basic structure of DNA

sugar/phosphate backbone and nitrogenous bases

Describe the basic structure of amino acids

amino group, side chain, carboxyl group

What is semi-conservative replication?

During replication, each new DNA molecuel has one strand from the parent and one new daughter strand.

Describe initiation of replication in bacteria

• initiation at single origin of replication (oriC) with IHF protein binding site

• unwinding begins at DUE (DNA unwinding element) site which is AT rich, done by helicase

• unwinds and starts replication forks

• 2 replication forks move out away from the origin until they have copied the whole region, helicase enzyme continues unwinding

• IHF protein helps stabilize

• DnaA protein (the initatior) binds to segments on OriC to repeat

Describe the helicase (DNAB) enzyme

• unwinds beginigng at DUE

• breaks h bonds and provides force for replisome

• 6 subunit ring, surrounds DNA

• requires ATP and proteins

Describe elongation in bacteria

• primase adds short RNA primer once template (parent strand) is free to make free 3’OH group on growing strand

• Tau connects DNA polymerase 3 to primosome (helicase and primase)

• DNA polymerase 3 adds nucelotides to the 3’ end of the RNA primer, building in 5’ to 3’ direction

• leading strand synthesis is continuous

• lagging strand is discontinous, short Okazaki fragments heading away from replication fork

• clamp/clc stabilizes DNA polymerase 3

• Topoisomerase relieves supercoiling

• SSB stabilizes strands

What are the roles of the all the proteins involved in DNA replication at the replication fork?

• helicase - unwinds

• primase - primes, synthesizes a complementary RNA primer

• DNA polymerase adds nucleotides with help from primase

• tau connects DNA polymerase 3 to primosome (heliase + primase_

• clamp - stabilized pol 3

• SSB - single strand binding proteins stabilizie strands

• topoisomerase - relieves supercoiling

How are lagging strands resolved?

• DNA polymerase 1 removes RNA primer, fills in gap with DNA

• DNA ligase links okazaki fragments, creating phosphodiester bonds

Describe lagging vs leading strand replication

• leading strand - synthesis is continuous with a single primer moving continuously towards replication fork

• lagging strand- discontinous synthesis, with multiple primers forming short fragments called Okazaki fragments, heading away from replication fork.

Describe DNA polymerase 3 proofreading

• after wrong H-bond formed, DNA pol stalls

• 3’ to 5’ exonuclease enzyme removes mismatch

• DNA poly resumes, fills in correct nucleotide

Describe DNA replication termination

topoisomerase induces DNA break to separate daughter chromosomes, then fixes them

Briefly describe the 3 steps of bacterial DNA replication

1. initiation - proceeds from origin of replication, DUE unwinds at A-T rich site. 2 forks move out in opposite directions. helicase continues unwinding

2. Elongation -primase adds RNA primer, DNA polymerase 3 extends strand in 5’ to 3’ direction, lagging and leading strand, clamp stabilizes, topoisomerase relieves super coiling

3. Termination - topoisomerase induces DNA break to separate daughter chromosomes then fixes them

Compare bacteria and eukarya DNA replication, inclduing

• origin

• replisome

• speed

• errors

• bacteria single origin, eukarya multiple origins

• bacteria fewer proteins at replisome, eukarya more complicated replisome

• bacteria faster than eukaryotes

• similar error rate

Does archaea replication more resemble bacteria or eukaryotes?

more resembles eukaryote, can have multiple origins

What is Taq polymerase?

• thermostable DNA polymerase

• discovered by Alice Chien

• used in original PCR techniaue

What is IHF in DNA?

• integration Host Factor

• DNA-binding protein that helps bring protein together

What is the role of the DnaA protein?

DnaA binds to OriC, bends the DNA, and helps initiate strand separation at the DUE.

What is the role of the RNA primer?

It provides a 3′-OH group for DNA polymerase to begin synthesis.

What enzyme seals the gaps between Okazaki fragments?

DNA ligase

What are the three key DNA elements at the bacterial origin of replication (OriC)?

• DUE – DNA Unwinding Element (AT-rich)

• IHF binding site – site for the DNA-bending protein IHF

• DnaA boxes – repeat sequences where initiator protein DnaA binds