Genetics Exam 1 (Rutgers Soliman)

characteristics of genetic material

1. must contain complex information

2. must be able to replicate

3. must encode the phenotype

4. must have the capacity to be different in different species

Fred Griffith's transformation experiment

Injected mice with strains of bacteria that either affected them or didn't, when the two were combined the mouse died.

1) injected with rough strain = mouse lives

2) injected with smooth strain = mouse dies

3)injected with boiled smooth strain = mouse lives

4) injected with rough strain and boiled smooth strain = mouse dies

Avery, MacLeod, McCarty

Determined that DNA was Griffith's "Transforming Factor."

~Boiled smooth strain was split into 3 samples. When Dna was destroyed, the animal survived

Protease

breaks down proteins

Rnase

breaks down RNA

Dnase

breaks down DNA

Hershey-Chase Experiment

Used radioactive phosphorous and sulfur to label DNA and protein respectively; infected bacteria passed on DNA; helped prove that DNA is genetic material not proteins

~T2 Phage are grown in mediums containing the radioactive material

Chargaff's Rule

A=T and G=C

Rosalind Franklin

Used X-ray diffraction to discovered:

1) the double-helical structure of DNA.

2)helix contains 10 nucleotides per turn

3) helix has a diameter of 20 angstroms (2nm)

Nucleotide

A building block of DNA, consisting of a five-carbon sugar covalently bonded to a nitrogenous base and a phosphate group.

Nucleoside

nitrogenous base + sugar

James Watson and Francis Crick

The scientists credited with building the first correct model of the structure of DNA by using Franklin's X-rays. They discovered:

1) DNA is double stranded and runs antiparallel

2) Bonds between A-T and C-G hold the 2 strands together

3) complementary basing suggests a mechanism for replication

Pyrimidines

Cytosine and Thymine

~has three rings

Purines

Adenine and Guanine

~ two rings

Nucleotides are joined by

phosphodiester linkages between the sugar of one nucleotide and the phosphate of the next

~do NOT break in heat

C + G

3 hydrogen bonds

A + T

2 hydrogen bonds

B form DNA configuration

the typical configuration in vivo---that proposed by Watson and Crick

~RIGHT handed helix

~found in cells

A form DNA configuration

under high salt concentrations, the helix gets a little more compact

~RIGHT hand helix

~ exists when DNA is isolated

Z form DNA configuration

seen in short DNA pieces with alternating purine/pyrimidine bases

~LEFT handed helix

tRNA (transfer RNA)

characterized by an anticodon, which binds the mRNA at the ribosome, and the amino acid-carrying portion

nucleoside analogs

mutagens that incorporate into DNA in place of a normal base; causes mistakes in base pairing

Conservative DNA replication

The original double strand serves as a new template for a new DNA molecule

Dispersive DNA replication

Both DNA molecules break down into fragment and serve as a template for the new fragment

~Both copies of genome are composed of scattered pieces of old and new DNA

semi-conservative DNA replication

The original strand unwinds and is used as a template to generate a new strand

Messelson-Stahl Experiment

Needed a way to distinguish the old from the new strand. They used two isotopes of Nitrogen: 14N and 15N

1) Grow the bacteria in heavy 15N for many generations. This made the DNA HEAVY

2) Took a sample of those bacteria and switched the rest to 14N. Took additional samples over a few cell cycles. New DNA contained 14N which was lighter

3) Ran the collected DNA on an equilibrium density gradient centrifugation to distinguish between heavy and light strands

Theta replication

Takes place in circular DNA such as that of E.coli

1) Double strand begins to unwind, producing single strands which function as a template for new strand. This is called the replication bubble

2) The unwinding continuous and the bubble get large. Location where there is unwinding is called the replication fork

3) DNA synthesis proceeds in the 5' to 3' direction

bidirectional replication

replication at both ends of a replication bubble

unidirectional

unwinding on one side

rolling circle replication

Takes place in some viruses and in the F-factor of the E.coli.

~NOT bidirectional

1) single strand breaks creating a 3' Oh and a 5' P group

2) new nucleotides are added on the 3' OH break using the inner strand as a template

3) as the new strand gets elongated, the original strand gets displaces, eventually rolling off the entire plasmid. This can continue for several rounds until many copies are made.

4)The linear fragment that rolls off can be used as a template for a new strand.

linear chromosomes replication

too large to have a single replication origin, so linear chromosomes in eukaryotes need multiple origins

Stages of DNA replication

initiation, unwinding, elongation, termination

Initiation (prokaryotic)

DnaA protein binds to the origin of replication (oriC)

~opens a small site for helices and single-strand binding protein to enter and begin unwinding the 2 strands

unwinding (prokaryotic)

DNA replication requires a single strand template. Multiple enzymes needed:

1. DNA helicase

2. Single-Strand binding protein

3. DNA gyrase

DNA helicase

breaks hydrogen bonds between DNA strands

single strand binding proteins

A protein that binds to the unpaired DNA strands during DNA replication, stabilizing them and holding them apart while they serve as templates for the synthesis of complementary strands of DNA.

DNA gyrase (topoisomerase)

prevents supercoiling and upstream torsional strain that builds up during unwinding

Topoisomerase I

creates single stranded breaks in DNA

Topoisomerase II

creates double stranded breaks in

~this is DNA gyrase

Elongation (prokaryotic)

single strands of DNA are used as a template to make a new strand. Need:

1. primase

2.primer

3. DNA polymerase

Primase

An enzyme that joins RNA nucleotides to make the primer.

primer

A short segment of DNA that acts as the starting point for a new strand

DNA polymerase III

replicates DNA using the RNA 3'-OH group

~has very high processivity: holds on to the DNA and doesn't release until its done

DNA polymerase I

removes the RNA primer and replaces it with DNA

Ligase

An enzyme that connects two fragments of DNA to make a single fragment

termination (prokaryotic)

some DNA replication terminate when the two replication forks meet

~others need assistance of termination protein Tus. The Tus protein binds to a sequence known as a Ter sequence. Upon binding, the complex blocks replication

leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.

lagging strand

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork.

differences between prokaryotic and eukaryotic DNA replication

origin

~prokaryotes typically have one origin. eukaryotes have multiple origins

differences between prokaryotic and eukaryotic DNA replication

cell cycle

~ prokaryotic DNA is replicating. Eukaryotic is controlled by the cell cycle

Licensing

takes place to ensure that replication fork initiate at the same time and doesn't initiate more than once per cycle

1. Origins are licensed. Licensing protein (origin recognition complex---ORC) get attached to the origin

2.Initiation machinery (MCM2-7 helicase) binds and starts replication. Licensing factor is removed as the replication fork moves away from origin. Licensing factor is removed to ensure that replication can't occur more than once per cycle.

Minichromosome maintenance (MCM)

has helicase activity and unwinds small stretch of DNA at the beginning of replication

differences between prokaryotic and eukaryotic DNA replication

DNA polymerase. 3 kinds:

1. Alpha: this has primate activity. Initiates DNA synthesis by generating a short primer

2. Delta: takes over after the primer has been laid down (by Alpha DNA polymerase) on the lagging strand

3. Epsilon: Functions like Delta but works on the leading strand

differences between prokaryotic and eukaryotic DNA replication

nucleosome

~eukaryotic DNA is complex and packaged for stability with histone proteins. Each nucleosome has 8 histone proteins, with DNA wrapped around it. During replication, the DNA needs to unwrap, and histones need to be removed so that all the DNA can be replicated

Step 1: Original nucleosome is removed

Step 2: some of the old histones find their way to their location on the new strand

Step 3: new histones are made and attach to the old histones

Telomere

repetitive DNA at the end of a eukaryotic chromosome

telomere shortening

chromosomes shorten every cell division. ~chromosome gets too short=apoptosis

Telomerase

An enzyme that catalyzes the lengthening of telomeres. The enzyme includes a molecule of RNA that serves as a template for new telomere segments.

replication

both strands are copied because there is a leading and lagging strand

transcription

one strand is copied.

template strand (non-coding)

strand that is copied

~also called antisense strand

non-template strand (coding strand)

The strand of DNA that is not transcribed during synthesis of RNA. Its sequence corresponds to that of the mRNA produced from the other strand.

~also called sense strand

~uses Uracil instead of thymines

transcription unit

A region of DNA that is transcribed into an RNA molecule.

~promotor, the RNA-coding sequence (gene), and the terminator

Initiation (bacterial transcription)

assembly of transcription machinery

1. machinery identifies promotor sequence

elongation (bacterial transcription)

DNA is made into RNA using RNA polymerase

~actually coding for mRNA from template DNA strand

termination (bacterial transcription)

separation of the RNA molecule from the parent DN strand

consensus

a promotor sequence that is recognized by the transcription machinery

~sits on exon-intron junction

Rho-dependent terminators

sequence in bacterial DNA that requires the presence of the rho factor protein to terminate transcription

Rho-dependent termination

1. RNA polymerase stops at terminator DNA

2. Rho attaches to a sequence on the RNA strand called the rho utilization site (rut). Rho binds to mRNA.

3. Rho then moves to the 3' end and unwinds the RNA from the DNA bringing transcription to an end

Rho-independent terminators

Based on an inverted repeat sequence that causes mRNA to create a secondary loop which forces the entire thing to fall apart

~destabilizes structure

Initiation (eukaryotic)

Nucleosomes

~The histones grip the DNA so tightly that the transcription factors cannot get in to bind with the promotor

~Acetylation of lysines in the histone proteins loosens the grip the histones have on the DNA

~Adding negatively charged acetyl groups to the lysin loosens the histones' hold on DNA

~this allows the transcription factors to bind to the promotor region

lysine

Positively charged R groups on histones

histone acetyltransferase (HAT)

any of a class of eukaryotic enzymes that loosen chromatin structure by adding acetyl groups to histone proteins

Histone deacetylases (HDAC)

-Remove acetyl groups

-Increases positive charges on histone tail (represses genes)

RNA polymerase I

present in all eukaryotes; transcribes large rRNAs

RNA polymerase II

present in all eukaryotes; transcribes pre-mRNA, some snRNAs, snoRNAs, some miRNAs

RNA polymerase III

present in all eukaryotes; transcribes tRNAs, small rRNAs, some snRNAs, and some miRNAs

Eukaryotic RNA polymerases

3 different polymerases recognize three different promotors

~all have different consensus sequences

initiation after nucleosomes (eukaryotic)

RNA polymerase II assembles on the promotor with another 50 polypeptides. This creates an open bubble for transcription to begin

elongation (eukaryotic)

doesn't start immediately

~about 30 bps are generated before polymerase enters the elongation phase

termination (eukaryotic)

Termination depends on the polymerase used

1. Poly I

2. Poly 2

3. Poly 3

polymerase I

uses a protein thats binds to the downstream DNA

polymerase II

can continue to work for 100s of nucleotides past its stop. Stopped by Rat1 or Xrn1.

polymerase III

transcribes a long stretch of uracils

Rat1

exonuclease that degrades mRNA being produced by RNA polymerase from 5' to 3' end, and when it catches up with the RNA polymerase, it causes the polymerase to fall off the template --> termination of transcription

XRN1

exonuclease that targets mRNA without methyl-G cap, degrades from 5' end

prokaryotic gene organization

prokaryotic DNA is colinear

~direct relationship between # of nucleotides and amino acids

eukaryotic gene organization

~contains exons and introns

~direct relationship between intron size and organism complexity-----> more complex=longer introns

Exons

Coding segments of eukaryotic DNA.

Introns

Noncoding segments of nucleic acid that lie between coding sequences and are spliced out.

RNA structure in prokaryotes

has a sequence called Shine-Dalgarno sequence which stabilizes ribosome

Shine-Dalgarno sequence

The prokaryotic ribosome-binding site on mRNA, found 10 nucleotides 5' to the start codon.

~sits separate from AUG

prokaryotic RNA processing

not much modification because transcription and translation happen simultaneously

~linked geographically and molecularly

~no processing of mRNA

Eukaryotic RNA processing

~5' Cap

~Poly A tail

~splicing

5' cap

a mehtylated guanine cap is added onto the 5' end of a mRNA molecule

~provides stability from enzymatic activity

Polyadenylation

addition of a short sequence or a tail of adenine (poly A-tail) nucleotides to the 3' end of an mRNA molecule.

~requires polyadenylation signal which is a consensus signal

~stabilizes RNA----> allows RNA to stay in cytoplasm for a long time

Splicing

the process of removing introns and reconnecting exons in a pre-mRNA

spliceosome

A large complex made up of proteins and RNA molecules that splices RNA by interacting with the ends of an RNA intron, releasing the intron and joining the two adjacent exons.

~consensus sequence tells where to cut

alternative splicing

taking all the exons and linking them in different ways to create new proteins

Redundancy/degeneracy

The genetic code has redundancy due to the fact that two or more codons can specify the same amino acid. (This is known as degeneracy) there are 4^3 (64) possible codons

aminoacyl-tRNA synthetase

An enzyme that binds to anticodon sequence and links it to the proper amino acid.