Final Exam, Genetics/ Biol 2153 Final- Larkin

The wobble hypothesis

Francis Crick

Telomerase is only made in immortal cells: gametes and stem cells

Cancer cells often reactivate telomerase and often have longer telomeres

The absence of telomerase in most somatic cells may contribute to aging (telomeres become shorter = speed up aging)

Cells do not contain reverse translation so any modifications made to a protein will not cause an inherited modification in the genome

Watson and Crick confirmed the triplet code of DNA, theorized the existence of an adapter molecule (tRNA), and proposed the central dogma of molecular biology, that information travels from DNA to RNA to protein

Central dogma concept relates to the "no inheritance of acquired characteristics" concept (giraffes do not get their long necks from stretching-Lamarck idea)

Lamarck coined the term "biology"

Prokaryotic genes are transcribed and translated within the same cellular compartment (no nucleus)

eukaryotic genes are transcribed and translated in separate compartments separated by the nuclear membrane

Eukaryotic gene expression involves additional RNA processing (RNA splicing)

snRNA, rRNA, tRNA, and mRNA are all encoded by gene and are all transcribed

the template strand or noncoding is the complementary strand of the sequence that will be in the RNA

The nontemplate or coding strand matches the Sense RNA strand

Difference between RNA polymerase and DNA polymerase:

-RNA polymerase uses NTPs (ribonucleotides) not dNTP's

-RNA polymerase does not need a primer

RNA polymerase creates a bubble of unwound DNA

5' prime end of mRNA is made first

Initiation (transcription) is the step where most regulation occurs

the DNA sequences required for initiation of transcription

promoter

prokaryotic transcription require -10 sequence and -35 sequence

sigma factor protein binds to the minus -35 sequence and directs RNA polymerase to recognize -10 sequence to start transcription

fewer hydrogen bonds (A and T regions) make it easy to pull apart (-10 sequence)

RNA polymerase adds first nucleotide at +1

Specificity of promoters is due to sequence-specific DNA binding proteins (transcription factors)

Eukaryotic transcription initiation requires basal transcription factors and regulatory transcription factors

eukaryotic promoter for transcription is the TATA box at position -25

other binding sites in eukaryotic transcription are CAAT, GC, and octamer boxes

TATA binding protein (TBP) is a component of basal transcription factor called TFIID that binds the TATA box and plays crucial role in assembling the collection of basal transcription factor to attract RNA polymerase

HIV life cycle

Attachment/Binding (CCR5 antagonist) - maraviroc

Fusion (fusion inhibitors) -Enfuvirtide (not widely used)

Reverse Transcription

-NRTI - Abacavir, didanosine, emtricitabine, lamivudine, stavudine, tonofovir, zalcitabine, zidovudine

-NNRTI - delavirdine, efavirenz, nevirapine, etravirine, rilpivirine

Integration (Integrase Inhibitors -gravir (raltegravir, dolutegravir, elvitegravir))

Replication

Assembly

Budding (Protease Inhibitors -Atazanavir, Fosamprenavir, Indinavir, Lopinavir/Ritonavir, Nelfinavir, Saquinavir, Tipranavir, Darunavir)

HIV

single stranded RNA genome

a lipid bilayer from its host cell (stolen from host)

reverse transcriptase

Watson and Crick

Developed the double helix model of DNA.

DNA

A complex molecule containing the genetic information that makes up the chromosomes.

anti parallel strands and base pairing

5 > 3

space filling model of DNA

Watson and cricks contributions:

1. double helix model

2. H bonded base pairing

3. DNA strands antiparallel

Roslind Franklin discovered:

the helix structure of DNA via X-ray diffraction

1. helical structure

2. distance between bases = 0.34 nm

3. repeat distance = 3.4 nm

genome size

The amount of DNA contained in one copy of a genome

eukaryotes have larger genomes than prokaryotes

-E. coli = 1 mm long genome

-Human genome = 2,000 mm long (diploid) 2 meters

eukaryotes need greater genome compaction than prokaryotes

composition of chromatin

DNA and protein

the material eukaryotic chromosomes are composed of

DNA from individual yeast chromosomes separated by _____

gel electrophoresis

each chromosome contains a single specific linear DNA molecule

Eukaryotic dna

unwinds in multiple areas as DNA is replicated

linear

DNA in super coils around nucleosomes

Nucleosome

repeating subunit of chromatin fibers, consisting of DNA coiled around histones

nucleosome core

4 histones (H2A, H2B, H3, H4) - 2 copies of each

and in an incomplete nucleosome = Histone H1

146 bp of DNA

Human metaphase chromosome

30 nm fivers are cold of nucleosomes

giving many levels of compacting DNA into chromosomes.

Nucleus found to contain

lots of large, acidic polymer rich in P, N, and carbohydrate- DNA

yet, seemed less structurally complex than protein, and most believed protein was the genetic material

F. Griffith

discovered genetic transformation in Streptococcus pneumonia

something in heat-killed (dead) bacteria could change phenotype of live bacteria

Oswald Avrey

set out to identify this "transforming substance"

Avery, MacCleod, and McCarty:

DNA is the transforming substance, not protein or RNA, & therefore DNA is probably the genetic material

When was bacteria and their viruses (bacteriophage) used (started to be used) in genetics

in the 1940s and 50s

life cycle of bacteriophage

entry of the nucleic acid causes the cessation of host cell DNA replication and protein synthesis

the host cell machinery is used for viral replication and synthesis of viral proteins

spontaneously assembly

cell lysis

Early in infection: virus takes over cell

Late in infection: production of new virus particles

Martha Chase and Alfred Hershey

discovered that DNA is the hereditary material in viruses

Provided more evidence DNA was the genetic material

Hershey-Chase Experiment

determined that DNA is the genetic material

Based on:

DNA has phosphorous but no sulfur

protein has sulfur, but try little phosphorous

Protein was labelled with Sulfur while DNA was labeled with Phosphorous

Only DNA gets into cell to replicate

human immunodeficiency virus (HIV), an RNA virus

Lipid bilayer

Reverse transcriptase in core

RNA genomes

the HIV life cycle

1. virus attaches to CD4 receptor (and other receptors) on hist cell by gp 20

2. viral RNA genome is transcribed into DNA by reverse transcriptase

3. Viral DNA is integrated into host genome

4. Integrated viral genome is transcribed into HIV genomic RNA and HIV mRNA

5. HIV particles are assembled and released from the cell by budding

note on the HIV lifecycle

1. reverse transcription of RNA genome --> DNA

2. Integration of viral DNA into host genome

3. Production of viral mRNA, proteins, and viral RNA genome

4. Assembly of mature virus and budding from cell surface

Nucleic acids have three main components:

1. phosphate

2. 5 carbon sugar (ribose or deoxyribose)

3. nitrogenous base

Structures of four DNA bases

know all the structures

RNA vs. DNA

ribose vs. deoxyribose, single stranded vs. double stranded, uracil vs. thymine

Structure of a Polynucleotide Chain

1. phosphodiaster linkage

2. 5' & 3' ends

Watson and Crick used 2 main lines of evidence to work out DNA structure

1. Erwin Chargaff: base ratios A=T, G=C

2. Roslind Franklin and Maurice Wilkins: X-ray diffraction of DNA fibers shows helical structure

Antiparallel strands and base pairing

strands run in opposite direction

base pairing by H-bonds

Space filling model of DNA

Watson & Crick's contributions:

1. double helix model

2. H-bonded base pairing

3. DNA strands antitparalles

5' --> 3'

3'<-- 5'

Genome sizes of organisms

Eukaryotes have larger genomes than prokaryotes

E coli genome

Human Genome

1mm long

2000 mm long (diploid (TWO meters!!)

Eukaryotes need greater genome compaction than prokaryotes!

Composition of chromatin

chromatin: the material eukaryotic chromosomes are composed of

Eukaryotic DNA is packaged into particles called

nucleosomes

DNA in supercoils around nucleosomes

Nucleosome structure

DNA is outside and histones are mainly inside

Core- four histones (H2A, H2B, H3, H4- 2 copies each)

146 bp of DNA

in an incomplete nucleosome: Histone H1

eukaryotic gene expression

5' and 3'

The tails of histones in nucleotides are subject to reversible covalent modifications

methylation of histone tails tends to lead to closed chromatin and tightly compacted nucleosomes that are harder to transcribe and is correlated to shutting off, decreased transcription

acetylation and phosphorylation of histone tails tends to lead to open chromatin and larger gaps lead to easier, increased to transcribe

transcription factors play a role in regulating chromatin

in prokaryotes, translation is occurring at the same time as mRNA transcription

prokaryotes diagram

eukaryotic modification post transcription:

1.) 7-MG Cap is added to the 5' end. 5' cap (methyG, linked to 5' to 5')

2.) Poly (a) tail is added to 3' end

3.) removal of introns (rna splicing)

Introns interrupt, but do not

scramble the DNA sequence that encodes a polypeptide

eukaryotic genes are interrupted by introns

if introns are not removed than the correct protein would not be produced

amino terminus (N-terminus)

carboxy terminus (C-terminus)

amino acids with hydrophobic (nonpolar) side chains: drives protein folding

Glycine, L-Alanine, L-Valine, L-Leucine, L-Isoleucine, L-Proline, L-Phenylalanine, L-Tryptophan, L-Cysteine

amino acids with hydrophilic (polar) side chains:

L-Serine, L-Threonine, L-Tyrosine, L-Asparagine, L-Glutamine

Amino acids with acidic side chains

aspartic acid, L-glutamic acid

Amino acids with basic side chains

L-lysine, L-arginine, L-histidine

Gene DNA sequence determine protein sequence so a single nucleotide change from a T could change a normal beta globin to a sickle cell globin substituting a glutamic amino acid that is hydrophobic (no water)

Francis Crick tried to figure out how mRNA could direct the synthesis of proteins. (tRNAs)

tRNAs act as an adaptor between nucleic acids and amino acids

Anticodon of tRNA base-pairs with codon in mRNA, in antiparallel orientation

3' end of each tRNA is charged with a specific amino acid by aminoacyl-tRNA synthetase

There are one to four different tRNAs per amino acid

there is an aminoacyl-tRNA synthetase for each tRNA

charging of a tRNA with the correct amino acid is essential for the genetic code to work

In eukaryotic translation, ribosomes (40S subunit and methionin) find the start codon by binding to the 5' cap and scanning from the 5' end of the mRNA to 3' until they find the first AUG start codon. Once that happens the 60S subunit joins to complete ribosome.

In prokaryotic translation, ribosomes recognize the start codon directly by binding to the Shine Delgarno sequence near the start codon

5'-to-3'; N terminus to C terminus

mRNA is synthesized in the ______ direction, which corresponds to the ______ of the protein.

Methionine is at the N terminus of the amino acid

Phenylaniline is at the C terminus of the amino acid

translation elongation:

1. insertion of aminoacyl-tRNA at the A site

2. Requires GTP and elongation factors

3. peptide bond formation catalyzed by 23S rRNA (growing peptide bond transfers from site P to A site

4. translocation of ribosome requires elongation factors

The Genetic Code:

1) Nucleotide triplets (codons) code for individual amino acids

2) Code is degenerate, more than one codon per amino acid

3) Code is ordered, codons for chemically similar amino acids often similar in sequence

4) Code is nearly universal (minor changes in mitochondrial code)

the occurrence of more than one codon per amino acid is called

degeneracy

occurs when the third base may be either of the two pyridines (U or C) or either of the two purines (A or G)

partial degeneracy

any of the four bases may be present at the third position in the codon and the codon will still specify the same amino acid (ex: Valine is encoded by GUU, GUC, GUA, and GUG

complete degeneracy

order in the genetic code has evolved as a way of minimizing mutational lethality

occurs when the first two nucleotides in mRNA codon are sufficient to determine the amino acid in the polypeptide specified by the mRNA

complete degeneracy

If the G:C base pair occupies the third (3′) position of the triplet,

hydroxylamine will induce amino acid substitutions only in cases where the genetic code is NOT degenerate, that is, where the base present as the 3′ nucleotide of the codon determines its meaning. Only two codons are not degenerate at the 3′ position; they are 5′-AUG-3′ (methionine) and 5′-UGG-3′ (tryptophan)