MCB 701 Exam 2 FULL SET

Griffith's experiment

- used pneumonococci and infected mice

- infected mice with a deadly (live) and non-deadly (heat killed or non-virulent) virus

- mice with virulent virus: died

- mice w/heat treated or non-virulent virus: lived

- mice w/ a mix of dead virulent and non-virulent virus: died

After seeing that infected mice with a mix of dead virulent and non-virulent virus died, Griffith then

- took samples from those mice to culture

- found that the mice had living virulent strains of virus in the samples

Griffith's experiment concluded that

- something from the dead virulent virus can transform living cells to cause infection

- the concept of transformation

Transformation (of pathogens)

- components of pathogens are capable of using DNA to communicate genetic information for infection

- proteins are NOT hereditary material

Base pairs occurring in a 1:1 ratio with each other means

the amount of A = T

the amount of G = C

Structural features of DNA

- Double stranded

- anti-parallel

- complementary base paired

- major and minor grooves

Minor groove

- the more constricted groove in the double helix

- harder to gain access too; likely to contain genes that aren't actively/frequently transcribed

The minor groove is partially blocked by

deoxyribosyl moieties (this is likely what keeps it restricted/ compact

Major groove

- the more open groove in the double helix

- can more easily bind DNA-specific proteins to the DNA (NT specific)

AT regions/base pairings in DNA are important because

- AT regions melt and lower temps compared to GC regions

- allows DNA to be denatured and opened for other processes (ex. replication)

Why do AT regions melt at lower temperatures in comparison to GC regions in DNA?

- AT bps are connected by two H-bonds, and GC bps are connected by three H-bonds

- AT bps are easier to dissociate since they only have two H-bonds

TATA box (promoter)

- a region in the DNA specifically w/high accumulations of AT base pairing

- it is easily melted, which allows DNA to unwind for gene expression

Nuclear DNA

- the bulk of all DNA in the cell organized into chromosomes

- 46 chromosomes, 23 chromosome pairs

- chromosomes contain between 48-240 billion bps

A nucleotide (NT) consists of

- a 5 carbon sugar

- a phosphate group

- a nitrogenous base

DNA strands always run from

5'-3'

The backbone of DNA consists of

the phosphate group and the sugar (phosphate-sugar backbones)

Mitochondrial DNA

- a small circular genome found only in the mito

- encodes for a small number of mitochondrial specific proteins

Examples of mitochondrial proteins encoded by mitochondrial DNA

- NADH dehydrogenases

- cytochrome B

- oxidases and ATPases

The synthesis of mitochondrial proteins takes place in

the mitochondria, with similar replication machinery

Telomeres

nucleoprotein complexes that cap the end of 3' DNA to ensure cell viability after each replicative cycle

Telomeres are composed of

tandem repeats of G-rich oligonucleotides (TTAGGG)

telomerase

a ribonucleoprotein complex that elongates telomeres on the ends of DNA

RNA

a single stranded nucleic acid composed of ribose

RNA structures

- single stranded

- contains uracil instead of thyamine

Since RNA is single stranded, it cannot

self replicate (no template for self replication)

Primary, secondary, and tertiary structures of RNA

- primary: chain of NTs

- secondary: folded into functional unit (ex. tRNA)

- tertiary: multiple folded RNAs working together as a unit (ex. ribosomal RNA)

rRNA

- ribosomal RNA

- a large complex of RNA molecules that are the structural and functional components of ribosomes

Nucleotide functions (6)

- building blocks for DNA/RNA

- activated intermediates of glycogen and lipids

- 2ndary messengers in signal transduction

- energy currency for ATP and GTP

- enzymatic function (Ribozymes)

- structural components of co-factors (NAD/NADH+)

Purine and pyrimidine carbon ring structure

- Purines: 2 carbon rings

- Pyrimidines: 1 carbon ring

Nucleobase

the nitrogenous base all by itself

Nucleoside

the nitrogenous base (nucleobase) + a phosphate group

A nucleotide is a nucleoside with

one or more phosphate groups (PO-)

Examples of nucleosides

- NMP: nucleoside 5' monophosphate

- NDP: nucleoside 5' diphosphate

- NTP: nucleoside 5' triphosphate

Deoxyribose differs from ribose in that

the placement of an -OH or -H on the 2' carbon

Deoxyribose has a _________ on the 2' carbon

-H

(deoxy: no oxygen)

Ribose has a _________ on the 2' carbon

- OH

Since RNA has a -OH in its carbon sugar, it prevents

long double helix regions from forming

How do NTs function as metabolic intermediates?

- contain phosphate groups that allows them to be/become energetic intermediates

- can transfer Ps to do work or use Ps to join a growing strand

Metabolic "forms"/examples of metabolic NTs

- NAD+

- FAD

- Coenzyme A

NAD+, FAD, and coenzyme A are all formed by

ATP phosphate donation

NAD

- nicotinamide adenine dinucleotide

- a pyrimidine NT formed from niacin (vit. B)

NAD+ is metabolically involved in

- redox rxns

- carries of reducing equivalents of hydride (H-) transfer rxns

NAD+ is also as a

cofactor for redox enzymes

Purine synthesis

the process of synthesizing purine carbon sugars

Purine synthesis occurs via what two pathways?

- De Novo

- Salvage

De Novo purine synthesis

- process of taking raw materials and a lot of ATP to produce a purine carbon sugar

- energetically expensive (11 enzymes; 7 ATPs required)

Raw materials for De Novo purine synthesis

- CO2

- NEAA (Asp, Glu, Gly)

- folic acid derivatives

Steps of De Novo synthesis

- starts w/Ribose 5'-P (synthesized from the PPP: pentose phosphate pathway

- Purines start as PRPP (Purine Ribose Pentose Phosphate)

- go from Inosine monophosphate to Adenosine or Guanine monophosphate

- N9 is added to complete the pathway

De Novo synthesis pathway is most active during ___________ and ___________;

De Novo synthesis pathway is also active in ___________ and ___________ tissues

the S-phase of the cell cycle

bone marrow and cancer

The 1st committed step of the de novo pathway is

the addition of the N9

Salvage pathway of purine synthesis

- the process of acquiring purines from food sources/endogenous NTs from ingestion/the environment

Which purine synthesis pathway is the most preferred?

- the salvage pathway

- especially when there is a presence of free floating NTs

- De Novo synthesis is very energetically taxing, which is why the salvage pathway is most preferred

Gout

- a disorder of purine catabolism

- purines cannot be broken down, leading to increased concentrations of urate and uric acid in the blood, urine, and joints

On a cellular level, gout results from ____________

On a metabolic level, gout results from ___________

- massive necrosis of tissue with insufficient purine catabolism

- insufficient enzymatic activity to clear uric acid

Xanthine oxidase

an enzyme responsible for the production of uric acid as a product

Treatments for gout use

xanthine oxidase inhibitors

Lesch-Nyhan Syndrome

- X-linked disorder characterized caused by a lack of the HGPRT gene

- leads to gouty arthritis, neuropathologies, mental retardation and aggressive behaviors

The HGPRT gene is responsible for

the conversion of PRPP into inosine monophosphate

the lack of HGPRT in Lesch-Nyhan syndrome causes

- an over-accumulation of PRPP

- increased purine biosynthesis

- increased accumulation of uric acid

Drugs that target NTs and nucleic acid metabolism

- cancer drugs

- antiviral drugs (base, nucleoside, and NT analogues

- anti-folates

Base-analogs alter

the nucleobases

Nucleoside analogues alters

- the nucleobases

- the sugar component

NT analogues alter/contain

a nucleic acid alteration with a normal sugar and phosphate group

Histones

- protein subunits that come together to allow for DNA organization

- fully formed histones allow DNA to wrap around it to form the nucleosome

Nucleosomes

- organizational structure of DNA

- comprised of DNA wrapped around histone proteins

Chromatin

further condensation of nucleosomes into fibers

Chromatin is commonly referred to as

beads on a string

- beads: nucleosome

- string: DNA; regions of DNA not wrapped around nucleosome called linker DNA

Chromatin organizes DNA specifically so

the right genes/areas of DNA are accessible for transcription

the accessible/transcribable areas of chromatin are

the regions of DNA NOT wound around a histone

DNA compaction/organization from small scale to big scale:

DNA strand > DNA wrapped around histones > nucleosomes > coiled into chomatin > chromatin condensed into chromosome

(DNA > nucleosome > chromatin > chromosome)

Nucleases

enzymes that break down nucleic acids

In chromatin, nucleases target __________ for digestion

linker DNA

Nucleases can't digest the DNA in a nucleosome because

the histone unit protects DNA from digestion

Histone units can be digested/dissociated by

- putting DNA in high salt concentrations

- disrupts histone into individual subunits

The core histone is comprised of

4 subunits that form an octamer

Histone subunits

H2A

H2B

H3

H4

process of forming the histone octamer

1. H3 and H4 dimerize , H2a and H2b dimerize unit)

2. H3-H4 dimers form tetramer

3. H3-H4 tetramer binds to DNA

4. H2a-H2b dimer binds to H3-H4-DNA complex

The ________ term tails of the histone stick out when bound to DNA

the N term elongated tails

Histones can associate tightly with DNA because

- Histones are positively charged

- DNA is negatively charged

- H-bonds form b/w the two

When the DNA associates with the histone, what regions of the DNA are more favorable?

- the minor groove

- regions with AT, AA, TT dinucleotide regions in the minor groove

- allows the DNA in these regions to be closer together/more compressed

In other words, the minor groove and regions of the DNA with AT,TT,AA di-NTs are

the more energetically efficient/favorable option for histone association

Histone H1

- a histone subunit not apart of the core histone

- "linker histone": binds to the DNA wrapped around the core histone to lock it into place in the nucleosome

Structural states of the nucleosome

- tetrasome

- hexasome

Tetrasome nucleosome state

DNA wrapped around an H3-H4 tetramer (abt 80 bps)

Hexasome nucleosome state

DNA wrapped around an H3-H4 tetramer and 1 H2a-H2b heterodimer

Structural states of the nucleosome can lead to/allow

transient DNA breathing

Transient DNA breathing

- a transient state of the nucleosome where the last 10-20 penultimate bps of DNA dissociate from the histone octamer

- allows these sites to be exposed (proteins can bind, transcription can occur, ect.)

The canonical nucleosome is

a left handed supercoil

Other than the structural states of nucleosomes, nucleosome structure can vary with

- direction of handedness of the DNA coil (L vs R hand)

- stoichiometry

The chromatin filament itself if mainly comprised of the

interactions of the N-tails of neighboring histones interacting

The chromatin filaments specifically organize into what shape?

a solenoid structure (a spring)

Solenoid structures in chromatin filaments are stabilized head to tail by

H1 histone subunits

Chromatin filaments further organize into chromosomes by using the

nuclear scaffold

Nuclear scaffold

- non-histone proteins that assemble to form a scaffold for chromatin to bind to/wrap around

- the innermost core of the chromosome

Primary, secondary, and tertiary structure of chromatin

1': "ball and string" motif: individual nucleosomes

2': nucleosomes condensing into solenoid structures; binding of H1 subunits

3': condensation of nucleosome solenoids into chromatin fibers

What factors alter the primary structure of chromatin?

- spacing b/w nucleosomes (due to DNA sequence, remodeling enzymes, and protein binding)

- post-translational modification of histones

What factors alter the 2ndary/tertiary structure of chromatin?

- long range interactions b/w nucleosomes in 1' structure

- stabilization of via architectural proteins (ex. H1)

The 2' and 3' structure of chromatin is important because

Its structure is what makes DNA functional and accessible in a cell

In the form of a chromosome/chromatin filaments, DNA can be accessed by/due to

- spontaneous unwraping from nuclesomes (mainly experiments)

- transition states of the nucleosome

- dynamic DNA-protein interactions that are reversible and highly regulated

Chromatin can be categorized as

euchromatin

heterochromatin