MICR5831 L1: Genes and DNA Replication 7/21/25

Where is DNA found in bacterial cells?

Nucleoid region

What form does a bacterial DNA chromosome typically have?

Single circular

Is bacterial DNA typically single or double stranded?

Double stranded

What does it mean for DNA polymers to be antiparallel to each other?

-DNA strands are reverse complements

-5->3 in opposite directions

-Complementary base pairing

True or False: Each nucleotide pair is a single base pair (bp)

True

What is this?

-Torsion

-Twisted bacterial chormosomes

-Negatively supercoiled loops

-Topologically independent, random distribution of boundaries

What is this?

-Plectonemes

-Branched loops arranged in linear order

-Radiate from NAPs core

What is this?

-NAPs

-Nucleoid Associated Proteins

-Plectonemes radiate from the NAPs core

What are possible arrangements for the circular chromatin fibers in a bacterial cell?

-Ring formation

-Intertwined

What bacterial growth phase is this?

-Lag phase

-Bacteria are introduced into fresh media

-Do not immediately replicate

What bacterial growth phase is this?

-Log phase

Bacteria are actively dividing at the maximal rate

What bacterial growth phase is this?

-Stationary phase

Population growth ceases but cells remain viable

What bacterial growth phase is this?

-Death phase

Viability of cells declines as they die

What bacterial chromosome site is this?

-Start point for replication

Ori (origin of replication)

What bacterial chromosome site is this?

-End point for replication

Ter (Termination site)

Where are the ori and ter sites found in the bacterial chromosome?

At the poles of the cell

True or False: The bacterial chromosome is separate from the cytoplasmic membrane

False, it is physically attached

What is the first step in prokaryotic DNA replication?

DNA replication is initiated at the origin of replication (ori)

What happens during prokaryotic DNA replication after DNA replication is initiated at the origin of replication (ori)?

-Expansion phase

-Bacterial cell size is increased almost two-fold

What happens during prokaryotic DNA replication after the Expansion phase (bacterial cell size is increased almost two-fold)?

-Resolution of the two chromosomes

-Moved to the opposite poles of the cell by an actin-like cytoskeleton in the cytoplasm

What happens during prokaryotic DNA replication after the two chromosomes move to opposite poles via the cytoskeleton?

A septum of new cell wall material is synthesized to separate the two cells

What happens during prokaryotic DNA replication after a septum of new cell wall material is synthesized to separate the two cells?

The cells are released from one another

What happens during prokaryotic DNA replication after the cells are released from one another?

Each daughter cell receives an exact copy of the chromosomal DNA

True or False: DNA replication is fully conserved

False, it is semi-conserved

What is this?

-Isomerase aka DNA gyrase

Unwinds supercoiled DNA

What is this?

-Helicase

-Unzips DNA double helix

-Exposes nitrogenous bases

-Two replication forks are formed

What is this?

-SSB (Single-Stranded DNA binding proteins)

Keeps DNA strands separate/apart

How is DNA replicated?

5' -> 3'

What is this?

-DNA primase

-Synthesizes RNA primers from DNA

What is this?

-DNA Polymerase III

-Binds single-stranded DNA

-Recruits deoxyribonucleoside triphosphates

-Matches them to the template strand via complementary base pairing

What is responsible for catalyzing the phosphodiester bonds in DNA?

DNA Polymerase III

What is responsible for synthesizing Okazaki fragments from the DNA template strand?

DNA primase

What is responsible for extending Okazaki fragments from the 3' end?

DNA Polymerase III

What is responsible for removing RNA primers and filling in the gaps?

DNA Polymerase I

What is this?

-DNA ligase

Joins Okazaki fragments together after they have been extended by DNA Polymerase I

True or False: After DNA replication, each identical dsDNA copy will have its own strand of original DNA

True

What is this?

-Catenated

Intertwined/chain-linked copies of the circular chromosome

What is this?

-Topoisomerase

-Introduces double-stranded break into dsDNA

-Separates catenated chromosome circles

Where are the two dsDNA strands re-joined to create a dimer after being separated by topoisomerase?

At the two ter sites

What happens at the two ter sites after the two strands are rejoined?

-Homologous recombination

-Strand exchange between dsDNA pairs

What is formed as a result of the homologous recombination occurring at the ter site between dsDNA pairs?

Holliday junctions

What is this?

-FtsK-XerCD recombination machinery

Cleaves the Holliday junction

What is responsible for repairing the phosphodiester bonds in the backbone after the Holliday junctions have been cleaved?

DNA ligase

True or False: All DNA is transcribed to create mRNA

False, intergenic regions between genes may not be transcribed

True or False: All genes encode mRNA

False, some genes encode for transfer RNA (tRNA) and ribonucleic RNA (rRNA) which are never translated

What is this?

-Genes

-Working subunits of DNA containing a particular set of instructions

-Encode gene products (tRNA, rRNA, mRNA)

True or False: Genes are found on both strands of DNA

True

True or False: Genes generally overlap

False

What is this?

-Found between -35 box and -10 box

-Region where RNA Polymerase binds to initiate transcription

Promoter

What is this?

-Found within 5' UTR

-Region where ribosomes bind to initiate protein translation

Shine Dalgarno Sequence

What is this?

-Found within 3' UTR

-Initiated by Stop codon (TAA, TGA, TAG)

Transcription termination signal

What is this?

-Found between 5' UTR and 3' UTR

-mRNA that is translated into protein

Open reading frame

How many predominant NAPs bend or bridge DNA?

Six

True or False: DNA bending NAPs do not have the same concentration during growth phases

True

What NAPs is this?

-Dimers of HNS are packed side by side between two adjacent DNA strands

-Preferentially binds curved DNA

-Covers 1% chromosome

Histone-like nucleoid structuring protein (H-NS)

What DNA does H-NS prefer to bind?

Curved DNA

What NAPs is this?

-Sequence specific DNA binding protein

-Bends DNA 50-90 degrees

-Acts as a transcriptional activator

Factor for inversion stimulation (FIS)

What does FIS do in addition to bending DNA by 50-90 deg?

Transcription activation

Which NAPs promote DNA supercoiling? H-NS or FIS?

Factor for inversion stimulation (FIS)

What is the outcome of this?

-Inhibition of transcription

-Nutrient deprivation, stringent response

-Low stable RNA synthesis

-RNAP dispersed

-Weak interaction between DNA loops

-Nucleoid decondensed

What is the outcome of this?

-High growth rates

-High stable RNA synthesis

-Appearance of transcription factors/foci

-Strong interaction between DNA loops

-Nucleoid condensed

When would you expect the nucleoid to be the most relaxed with very little transcription?

Stationary phase

During what phase would you expect the nucleoid to be highly supercoiled and transcription factories to be created (for the purpose of producing rRNA)?

Log phase

True or False: mRNA is not spliced in prokaryotes, only in eukaryotes

True

True or False: Protein is not spliced in prokaryotes, only in eukaryotes

False, bacteria do some protein splicing

Describe the component parts of a nucleotide and how this forms a DNA strand. (slide 5 and 6)

1) Nucleotide

2) Nitrogenous bases

3) Phosphodiester bonds

4) Pentose sugar (Deoxyribose)

-Complementary nitrogenous bases will base pair with each other

-Hydrogen bonding, weak interaction

Using a diagram explain the structure of a DNA double helix. (slide 7)

-Two anti-parallel DNA polymers

-Each DNA strand is reverse complement of the other

-Runs 5->3 in opposite directions antiparallel

-Each nucleotide is 1 bp

What are the features of a bacterial chromosome? (slide 8)

-Bacterial chromosomes are circular with twisted (torsion)

-Nucleoid is condensed via negatively supercoiled DNA loops (topologically independent)

-Branched loops (plectonemes) arranged in linear order and radiate from a core of NAPs

-Circular chromatin fiber can be organized as a ring or intertwined

What are the important features of the chromosome for DNA replication (slide 10)

-Origin of replication (ori) is the only start point for replication

-Termination site (ter) is the end point for replication

-The chromosome is organised so that the ori and ter sites are found at the poles of the cell

-The chromosome is physically attached to the cytoplasmic membrane

What are the steps in DNA replication during cell division (Slide 11)

1) DNA replication is initiated at the origin of replication (ori)

2) Expansion phase where the bacterial cell size is increased almost two fold

3) Resolution of the two chromosomes which are moved to the opposite poles of the cell by an actin-like cytoskelton in the cytoplasm

4) A septum of new cell wall material is synthesized to separate the two cells

5) The cells are released from one another

Outcome: Each daughter cell receives an exact copy of the chromosomal DNA

Name two proteins and explain their function during the formation of the replication fork (Slide 12)

1) Isomerase (DNA gyrase) unwinds supercoil

2) Helicase unzips DNA double helix exposing nitrogenous bases, forming two rep

Name the enzyme and explain its function during the replication of the leading and lagging strands of DNA. (slide 12)

Leading Strand:

-DNAPol synthesizes DNA continuously in the direction of replication fork movement.

Lagging strand:

-DNAPol synthesizes DNA discontinuously in short fragments called Okazaki fragments, requiring multiple primers.

Explain the four steps involved in resolving a catenane (Slide 13)

1) A double stranded break is introduced into the dsDNA by topoisomerase

2) The strands are joined to create a dimer at the two ter sites and homologous recombination occurs at the ter to form Holliday junctions

3) The dimer is resolved by the FtsK-XerCD recombination machinery which cleaves the Holliday junction

4) DNA ligase repairs the phosphodiester bonds in the backbones to create an uninterrupted backbone

How are genes organized on bacterial chromosomes (Slide 15)

-Bacterial chromosomes are single, circular DNA molecules

-Organized into a compact structure within the nucleoid.

-Supercoiling and interactions with nucleoid-associated proteins (NAPs)

Draw a gene and label all the features necessary for expression (slide 16)

-Promoter

-5'UTR

-Translation start point +1

-Start translation codon ATG

-ORF of mRNA

-Stop codon TAA< TGA, TAG

-3' UTR

-Transcription termination signal

Describe the functions of two nucleoid associated proteins (slide 17).

1) Histone-like nucleoid structuring protein (H-NS)

-Dimers of HNS are packed side by side between two adjacent DNA strands

-Preferentially binds curved DNA

-Covers 1% chromosome

2) Factor for inversion stimulation (FIS)

-Sequence specific DNA binding protein

-Bends DNA 50-90 degrees

-Acts as a transcriptional activator

-Promotes DNA supercoiling

What are transcription factories? When and how are these formed? (Slide 18 and 19).

-Transcriptional and translational machinery form factories

-Large mega structures for the synthesis of RNA and translation of proteins

-Triggers: High growth rates, high stable RNA synthesis

How does chromosomal architecture change during nutrient deprivation and high growth rates (slide 19)

Nutrient Deprivation:

-RNAPol dispersed

-Weak interaction between DNA loops

-Nucleoid decondensed

High Growth Rates:

-Transcription factories and foci appearance

-Strong interaction between DNA loops

-Nucleoid condensed