BCH 4024 Exam 4

“Central Dogma”

A way to describe flow of genetic information in the cell

• DNA is replicated → DNA is transcribed into RNA → RNA is translated into protein

In prokaryotes, DNA is in the…; in eukaryotes, DNA is in the…

Cytoplasm; nucleus

In Streptococcus pneumoniae, smooth cells produce…, while rough cells…

Polysaccharide capsule; don’t produce this capsule because they don’t have genetic material to make it

What strain of Streptococcus pneumoniae causes pneumonia?

Smooth; because the capsule protects it from host’s immune system

Avery’s experiment

• Used smooth and rough strains of Streptococcus pneumoniae

• Relied on “bacterial transformation” technique

• Heated up smooth cells, causing DNA to be released into environment (cell free extract)

• Mixed modified (with proteinase, RNase, or DNase) cell free extract with living rough cells

• Rough cells took up smooth DNA and conditionally transformed into smooth cells

Bacterial transformation

External DNA is taken up by a bacterial cell, changing characteristics of the cells

First Avery’s Experiment

Treated smooth cell free extract with proteinase; rough cells still transformed into smooth cells

Second Avery’s Experiment

Treated smooth cell free extract with RNase; rough cells still transformed into smooth cells

Third Avery’s Experiment

Treated smooth cell free extract with DNase; rough cells DID NOT transform into smooth cells

What did Avery’s Experiments show?

DNA IS in fact the genetic information of the cell

Know nucleotide structures

Know nucleotide structures

Nucleotide

Nitrogenous base, sugar, and AT LEAST one phosphate

On ribose of nucleotides, what is on the 3’ and 2’ Cs, respectively

• 3’: OH

• 2’: OH in ribose (RNA), H in deoxyribose (DNA)

On ribose of nucleotides, what is on the 1’ C?

Glycosidic bond connecting ribose to nitrogenous base

How many rings do purines and pyrimidines have, respectively?

• Purines: 2

• Pyrimidines: 1

Nucleoside

Nucleotide minus the phosphate

What bond links nucleotides together?

Phosphodiester linkage

• between 3’ OH and 5’ phosphate

What direction are polynucleotide sequences written?

5’ → 3’

Chargaff’s rules

1. DNA has concentrations in which A=T and G=C

2. Base composition varies between species

3. Base composition the same in different cells within individual organism

4. Base composition does NOT change with age/nutrition/environment

Photo 51

• First x-ray diffraction image of DNA (by Rosalind Franklin)

• DNA is double stranded, helical, and reveals information about turns

Repeating unit of DNA

dNMP

DNA contains… chains

Two unbranched polynucleotide chains

DNA backbone

Sugar and phosphate portion

… hydrogen bond(s) between As and Ts, and… hydrogen bond(s) between Gs and Cs

2; 3 (Watson and Crick base pairing)

Sugar/phosphate backbone of DNA is connected via…

Phosphodiester bonds

What bonding connects base pairs?

Hydrogen bonds (non-covalent bonding)

What does dNMP stand for?

2’-deoxyribonucleoside 5’-monophosphate

Why AREN’T DNA strands bonded covalently?

Bonds need to be easily breakable/reversible to separate strands for replication

Separating two DNA strands is termed…

Denaturing

Tm (melting temperature)

Temperature at which a solution of DNA is 50% denatured

What DNA regions are higher in Tm?

GC-rich regions; more hydrogen bonds, more energy input to break

Annealing

When complementary single strands of DNA come together through base pairing (i.e., opposite of denaturing)

In a living cell, >99% of DNA is in… conformation

B-DNA

B-DNA conformation

1. Right-handed helix

2. 10.5 base pairs per turn of helix

3. Base pairs lying perpendicular to backbone

4. Hydrophobic portion of bases on interior

5. Base pairs exposed in major/minor grooves

Major and minor grooves

• Major: where a lot of nitrogenous bases exposed

• Minor: where less nitrogenous bases exposed

A-DNA

Conformation of dehydrated DNA

Z-DNA

Seen in GC-rich regions

RNA’s 2’-hydroxyl on ribose sugar makes it a more… molecule

Fragile; can break phosphodiester bonds

RNA sequences are always read from…

5’ → 3’ direction

G-U base pairing

Allowed when RNA base pairs with itself or another RNA molecule

What types of structures does RNA have, if any?

• Primary structure: sequence

• Secondary structure: base pairing within same RNA molecule (e.g., stem loops)

• Tertiary structure: interaction of various secondary structures

Gene

DNA sequence that encodes something functional

• Includes regulatory sequences

Genome

Complete set of genetic information of all genes within a cell

Chromosome

DNA molecule that encodes genes

In a genome, the positions of genes are… within a species

Fixed

What is special about the E. coli genome?

Single, circular

What types of chromosomes do humans have?

Linear chromosomes

In the nucleus, chromosomes are found…

Wrapped around proteins

Meier-Gorlin Syndrome

• DNA replication initiation impaired, from mutated helicase gene (MCM5)

• Small ears, no kneecaps, gaps in skull

Conservative replication

Yields one original DNA molecule and one entirely newly synthesized DNA

Semi-conservative replication

Yields two DNA molecules, each with one parental and one newly synthesized strand

Dispersive replication

Yields two DNA molecules that are hybrids (or mixtures) of parental and newly synthesized DNA 

Meselson and Stahl experiment

• All parental DNA labeled with ¹⁵N → saw single heavy density band

• Allowed to replicate in ¹⁴N medium → saw single hybrid density band

• Allowed to replicate again in ¹⁴N medium → saw one hybrid density band, and one light density band

• Concluded that DNA replication is semi-conservative

Meselson and Stahl used… cells

E. coli

Meselson and Stahl used… culture media

(1) regular culture media and (2) culture media with heavy isotope of N

Meselson and Stahl used… technique

CsCl density gradient

CsCl density gradient

If CsCl is put into centrifuge, a density gradient is created down tube (heaviest density at bottom); DNA will migrate to equivalent density

DNA polymerase substrate

dNTP

DNA polymerase

Catalyzes extension of DNA strand one dNMP at a time

DNA polymerase synthesizes in… direction

5’ → 3’

DNA polymerase catalytic mechanism

3’ hydroxyl of 3’ nucleotide attacks a phosphate of incoming dNTP, releasing PPi

What is required at active site of DNA polymerase?

Two Mg2+ ions; stabilizes charges to set up conditions for nucleophilic attack

DNA polymerase requires a… and…

Template; DNA or RNA primer

DNA polymerase replication is…

Accurate; avoids mutations

Nucleases: DNase is specific for…, while RNase is specific for…

DNA; RNA

Nucleases can…

Remove bases from DNA/RNA

Exonuclease

Breaks phosphodiester bond at one end of polynucleotide chain

Exonucleases can work in the… direction

5’ → 3’ OR 3’ → 5’

Endonuclease

Breaks a phosphodiester bond within a polynucleotide chain

Endonucleases can be… or…

Sequence-independent; -specific

Endonucleases can make… break(s)

Single-strand (nick) or double-strand

Excinuclease

Breaks two phosphodiester bonds within a single polynucleotide chain

Restriction endonucleases

Endonucleases that only break phosphodiester bonds at specific DNA sequences (restriction sites)

Properties of restriction sites

• Short

• Palindromic

• 4-6nt long

Palindromic sequences

Both strands of DNA have same sequence when read 5’ → 3’

• AATT and TTAA

High-fidelity DNA polymerases have…

Two active sites:

• Catalytic site for DNA synthesis

• 3’ → 5’ exonuclease site for removing mis-incorporated nucleotides (like a backspace button)

DNA synthesis begins at…

An origin of replication sequence

How is DNA synthesized starting at the origin?

In both directions; bidirectional synthesis

DNA is synthesized by DNA Pol at sites called…

Replication forks

For every origin of replication, there is(are)… replication fork(s)

2

Parent DNA is unwound by…

Helicase

DNA synthesis at the leading strand occurs…, while DNA synthesis at the lagging strand occurs…

Continuously; discontinuously

Okazaki fragments are synthesized by…

A series of discontinuous 5’ → 3’ reactions at the lagging strand

Supercoils

From under- or over-winding DNA

Topoisomerases

Enzymes that add/remove supercoils in DNA by cutting phosphodiester bonds → unwrapping helix → resealing strands

DNA gyrase

Bacteria-specific topoisomerase that introduces supercoils (in addition to removing them ahead of replication fork)

Why would DNA gyrase introduce supercoils?

To compress DNA for packaging

Fluoroquinolones

Antibiotics that target bacterial DNA gyrase

Fluroquinolones block…

Ability to reseal DNA, causing double-strand breaks

Fluoroquinolones are effective in fighting bacterial infections because they have…

Selective toxicity; DNA gyrase is not present in humans and therefore our cells are not threatened by fluoroquinolones

What is the regulated step in the control of DNA synthesis?

Initiation

oriC

Origin of replication in E. coli; a unique 245 bp sequence

DnaA protein

First protein that comes in for DNA replication in E. coli; binds to R and I sites

DNA unwinding element (DUE)

AT-rich segment where strand separation occurs

Why does DUE bind to an AT-rich region?

In these regions, DNA strands bound using 2 H bonds only, requiring less energy/making it easier to separate them

How is DnaA activated?

Binds ATP

DnaA proteins bind to oriC →

Positive supercoil → DNA denaturation at DUE → replication bubble formed

What does DnaC doe when it binds to ATP?

Loads a DnaB helicase at both ends of replication bubble

Why is it important that DnaA is very slow to release ADP after dissociating?

It prevents dissociated DnaA from binding ATP and initiating replication at the same origin as before (each origin can only be used once)

Dam methylase

Methylates oriC DNA to regulate initiation