Review Set 2 Flashcards
DNA Structure
DNA is a nucleic acid made of nucleotides; 4 of them in DNA, each with a different base
Each nucleotide is made of a phosphate group covalently bonded to a pentose sugar
Nitrogenous Bases in DNA
Adenine, Thymine, Cytosine and Guanine
How are DNA Nucleotides grouped together?
by phosphodiester bonds in a 5’ to 3’ direction to form a single strand
How is double stranded DNA created?
created when hydrogen bonds form between bases (A + T and C + G) between single strands
Double stranded DNA are….
Antiparallel
Rosalind Franklin and Maurice Wilkins at King’s College (DNA structure using x-ray diffraction) contributions to DNA Structure understanding
X-ray diffraction/ X-ray crystallography using crystallized DNA molecules X-ray beams pass through crystallized DNA (for tens of hours) and diffract (spread) when they hit atoms (or other objects) and their scattering pattern is recorded on a special film The scattering pattern produces an image from which a 3D structure can be deduced
James Watson and Francis Crick at Cavendish Laboratory in Cambridge contribution to Discovery of DNA
Molecular models of DNA using all evidence available
PHOTO 51 importance
DNA is a double helix
Phosphate groups on outside of molecule (backbone)
Nitrogenous bases on inside of molecule
Nucleosome
Fundamental unit of DNA packaging – allows supercoiling of DNA into chromosomes
Core = 8 histone proteins (+ charged) with DNA molecule (- charged) wrapped twice around
Different types of sequences for DNA
- Unique (single-copy) sequences = genes (code for proteins)
-2% of genome - Highly repetitive sequences = found
between genes (form barriers of non-
coding regions between genes)
-5 to 45% of genome
-Short-tandem repeats (STR’s): form
polymorphisms (significant variation
between individuals – used to create
DNA profiles)
-Transposable (moveable = shuffle
genes) - Structural Sequences = pseudogenes (highly coiled at centromeres and telomeres)
-20% of genome
Hershey- Chase Experiments
Used bacteriophages (viruses that infect bacterial cells – made up of DNA and a protein coat) with radioisotopes (radioactive forms of elements that decay at a predictable rate – can detect these in cells)
Used radioactive phosphorus and radioactive sulfur
Phosphorus found in DNA (phosphate groups)
Sulfur found in proteins
Created one type of bacteriophage with radioactive phosphorus and another type with radioactive sulfur
Allowed two different types of phages to infect bacterial cells
RESULTS:
Bacterial cells infected with radioactive phosphorus produced new phages with radioactive DNA.
Bacterial cells infected with non-radioactive phosphorus produced new phages with non-radioactive DNA.
None of the new viruses had radioactive sulfur (radioactive phosphorus was found in the pellet)
DNA was passed on to the new viruses, and protein was NOT!
Protein is NOT the genetic material and DNA is!
DNA REPLICATION
- Helicase unzips the parental DNA molecule (breaking H-bonds between bases)
Note: in eukaryotes, gyrase and single-strand binding proteins stabilizes unzipped DNA molecules at many sites - Primase adds a sequence of RNA bases (a primer) to each parental DNA molecule at the replication origin (each parental molecule serves as a template)
- DNA polymerase III adds new nucleotides (deoxynucleoside triphosphates – two phosphates lost to provide energy for binding) to the RNA primer (at the 3’ end ONLY) to create a new complementary strand (one for each of the parent DNA molecules – A binds to T, C binds to G) Continuous in the leading strand, as Okazaki fragments in the lagging strand (moves in a 5’ to 3’ direction – adding new nucleotides to the 3’ end only!)
- In the lagging strand, DNA ligase fills the gaps between fragments (5’ to 3’)
- DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides ( 5’ to 3’ direction – DNA bases left unpaired at the tip of the 5’ end after primers removed)
What does Helicase do?
unzips the parental DNA molecule (breaking H-bonds between bases)
What does Primase do?
Adds a sequence of RNA bases (a primer) to each parental DNA molecule at the replication origin
What does DNA polymerase III do?
Adds new nucleotides (deoxynucleoside triphosphates – two phosphates lost to provide energy for binding) to the RNA primer (at the 3’ end ONLY) to create a new complementary strand (one for each of the parent DNA molecules – A binds to T, C binds to G) Continuous in the leading strand, as Okazaki fragments in the lagging strand (moves in a 5’ to 3’ direction – adding new nucleotides to the 3’ end only!)
What does DNA Polymerase I do?
removes the RNA primers and replaces them with DNA nucleotides ( 5’ to 3’ direction – DNA bases left unpaired at the tip of the 5’ end after primers removed)
Meselson and Stahl Experiments
Used 2 different isotopes of nitrogen to grow bacteria (E. coli) cells (14N and 15N)
First, cultured/ grew bacterial cells in medium containing 15N (which is heavier than 14N)
After many generations, all bacterial cells contained 15N in their DNA
15N bacteria transferred to medium containing 14N
After 1 generation in 14N medium, bacteria removed and DNA isolated
Dissolved DNA in solution and centrifuged (spun around very quickly – this separates dissolved contents based on their density – more dense items sink lower in the tube, lighter items stay closer to top of tube)
14N DNA is light, so it would be found at top of tube; 15N DNA is heavy, so it would be found at bottom of tube
Results: ALL DNA in F1 (first) generation made up of one strand with 14N and one strand with 15N (all found in middle of test tube) – this shows that DNA replication IS semiconservative!
Genes are made up of…
are made up of specific sequences of nucleotides that “code” for the sequence/order of amino acids that are put together (by ribosomes) to make up each protein
DNA Sequences Determine?
The amino acid sequence of EVERY protein in a cell (its primary structure), which influences every level of protein structure after that, which determines the protein’s overall structure, which affects its ability to function properly
Process of making Proteins
DNA –Transcription–> RNA –Translation–> Protein
What is Transcription?
RNA Polymerase (and transcription factors = proteins in eukaryotes) reads genes (antisense/ template strand of DNA) and makes mRNA molecule based on code in DNA RNA is a nucleic acid (nucleotides)
Translation
making a polypeptide chain – a protein - (putting amino acids together) from mRNA
Compare DNA and RNA
DNA, RNA
Double stranded, single stranded
Deoxyribose, Ribose
Guanine, Cytosine, Adenine, Thymine (bases) and Guanine, Cytosine, Adenine, Uracil
Steps of Transcription
- Initiation
- Elongation
- Termination
Initiation- Transcription
RNA polymerase unwinds DNA strands and binds to promoter on DNA
Elongation transcription
RNA polymerase adds RNA nucleotides (nucleoside triphosphates – two phosphates lost to provide energy for binding) to 3’ end of growing mRNA strand based on code in antisense strand of DNA
Works in 5’ to 3’ direction
Bases added using complementary base pairing rules
Termination transcription
RNA polymerase continues until terminator sequence reached (passes in eukaryotes)
mRNA molecule detaches from DNA
RNA polymerase detaches from DNA
Ribosome structure
rRNA (nucleolus) and proteins
Small subunit (with mRNA binding site) – binds to large subunit ONLY during translation
Large subunit (with tRNA binding sites – A site, P site, E site)
Form polysomes (several ribosomes translating mRNA at the same time)
70S (density) in prokaryotes; 80S in eukaryotes
Free ribosomes synthesize proteins for use in the cell
Bound (RER) ribosomes make proteins for secretion/ use in lysosomes
tRNA faccilitates
translation
Things you need to know about the Genetic code
Translation occurs following the genetic code mRNA molecules contain codons(series of 3 – triplet – bases) Each codon specifies ONE amino acid (61 of 64) Start codon (AUG) specifies methionine Stop codons (3 of them -do not code for amino acids – end translation) Code is universal to ALL life!
Steps in translation
Initiation
Elongation (and translocation)
Termination
Initiation
Small subunit of ribosome binds to mRNA at start codon (AUG)
tRNA (with complementary anticodon UAC) binds to mRNA (complementary base pairing)
tRNA carrying amino acid methionine
Large subunit binds (with 1st tRNA in P site) - GTP
Elongation (and translocation)
2nd tRNA comes into A site (complementary base pairing with mRNA codon) Peptide bond forms between amino acids of two tRNA molecules
1st tRNA moves (translocates) into E (exit) site and leaves ribosome
2nd tRNA moves (translocates) into P (polypeptide) site
Ribosome moves along mRNA in 5’ to 3’ direction
3rd tRNA comes into A site
Peptide bond forms between amino acids of two tRNA molecules (one in P site and one in A site)
And so on… until
Termination
Stop codon (on mRNA) reached
Polypeptide chain released from tRNA in P site
Ribosome disassembles
What is gene expression?
Cells become different (differentiate) because they have different genes “turned on”, thus they make different proteins
Genes that are “turned on” are being expressed in a cell (they are actively being used to make mRNA, which will be used to make proteins)
Gene expression is affected by an organisms…
epigenome
Epi = above, genome = entire collection of DNA sequences (“above the genome”)
Epigenome = a collection of all the factors that modify/ impact the activity/ expression of genes without altering DNA sequences
Things that effect gene expression
- Nucleosomes
More nucleosomes = DNA packaged more tightly together/ genes less accessible to RNA polymerase (less transcription/ less mRNA/ less protein from those genes, if any at all) - Methylation
Methyl groups (CH3) bind to DNA, causing it to wrap more tightly around histones
More methylation = less transcription/ less protein from those genes (if any)
Highly methylated genes are usually not expressed at all, and methylation of DNA is maintained through cell division and even from parent to offspring! - Proteins
Transcription factors – aid in RNA polymerase binding to DNA
Transcription activators/ transcription repressors - The Environment
Can change methylation patterns and/ or affect proteins involved in regulating gene expression/ splicing (wrong genes on or off etc.)
Protiens
Proteins made up of amino acids that are put together in a specific order based on the sequences of nucleotides in DNA
The entire collection of proteins in an individual (or in one of its cell types) is called its proteome
Because proteins are put together based on DNA, each individual has a unique proteome, as well as genome
Primary level of protein structure
Sequence (and number) of amino acids
Amino acids linked by peptide bonds
Secondary level of protein structure
Folding pattern (basic) of polypeptide Two types: Alpha helix (keratin in hair) / Beta-pleated sheets (spider silk) Held by hydrogen bonds/ stabilizes structure of fibrous proteins Interactions between amino and carboxyl groups
Tertiary level of protein structure
Folding pattern of polypeptide into 3D shape (for function/ active site if an enzyme)/ globular proteins
Stabilized by disulfide bridges, ionic bonds, hydrogen bonds, hydrophobic (Van der Waals) interactions
Interactions between R groups
Quaternary level of protein structure
Not in all proteins
Linking several polypeptide chains together (using same bonding as tertiary structure)
Linking prosthetic group to polypeptide
Polar proteins
form inner portions and cytoplasm/ extracellular portions of membrane proteins (hydrophilic channels through cell membranes); form active sites on enzymes (attract polar substrates); allow proteins to dissolve in water
Non polar proteins
form outer portions of membrane proteins (toward phospholipid tails) and proteins embedded in cell membrane; form active sites on enzymes
Shape determines
Function
Enzymes/ catalysts
catalase, amylase, lipase, polymerase
Movement
actin, myosin
Structure
collagen, elastin, keratin
Transport
hemoglobin
Defense
immunoglobin, antibodies
Hormonal communication
insulin, luteinizing hormone
Fibrous vs Globular protein
Fibrous vs Globular
Long, narrow vs Rounded, spherical
Repetitive (no 3° structure) vs Irregular
Structure/ support vs Enzymes/ metabolism
Insoluble (in water) vs Soluble (in water)
Actin, myosin, keratin, collagen vs Hemoglobin, insulin, amylase
Enzymes are
Catalysts
Enzymes are specific to their
Enzymes are specific to their substrates: active site (place where substrate binds) has specific shape for substrate (like lock and key) makes/ breaks bonds in substrate while holding it in optimum position binding of substrate to active site forms enzyme-substrate complex; causes conformational (shape) change in active site (in R groups of amino acids) to “fit” substrate better/ make substrate more reactive (induced fit)
What effect enzyme activity
Temperature: at optimum enzyme works best/ fastest
(more collisions between molecules as temp. increases);
below rate slows down; above enzymes denature (lose
characteristic shape – permanent if peptide bonds break)
pH: at optimum enzyme works best/ fastest; below/
above enzymes denature (ions interact with active site/
ionic/ hydrogen bonds)
Substrate concentration: enzyme activity increases as
substrate concentration increases up to a point where
enzyme activity plateaus – enzymes saturated (working
as fast as can – all active sites occupied)
Anabolic
building” of complex molecules, endergonic – more energy IN to build bonds, often involves condensation, biosynthetic
Catabolic
“breaking down” of complex molecules, exergonic – more energy released as chemical bonds are broken, often involves hydrolysis, degradative/ digestive
Different types of inhibotors and compare
Competitive vs Noncompetitive
Shape is similar to substrate vs Shape is NOT similar to substrate
Binds to active site; blocks active site and prevents substrate binding vs Binds to other site (allosteric site); causes change in active site shape so substrate cannot bind
Increase in substrate concentration reduces inhibition vs Increase in substrate concentration does NOT reduce inhibition
Reversible inhibition vs Irreversible inhibition (USUALLY)
Metabolic pathways are regulated by
end-product inhibition
End product inhibition is
Negative feedback mechanism
Enzymes in The real world
Lactase (lactose glucose + galactose) for lactose intolerant individuals
Obtained from yeast/ bacteria
Pre-digest lactose in milk/ dairy products using immobilized enzyme (trapped in calcium alginate beads)
Makes products sweeter too (without added sugar)
