DNA, RNA, Proteins, and Enzymes (Review #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 (deoxyribose) bonded to a nitrogenous base)
-Nitrogenous bases in DNA: Adenine, Thymine, Cytosine and Guanine
5’ to 3’ binding overview
(Binding nucleotides together in the 5’ to 3’ direction – condensation – removal of water and formation of covalent bonds called phosphodiester bonds – nucleotides can ONLY be added to the 3’ end!!!)
Rosalind Franklin and Maurice Wilkins at King’s College (DNA structure using x-ray diffraction)
-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
Photo 51 by Rosalind Franklin
-DNA is a double helix
-Phosphate groups on outside of molecule (backbone)
-Nitrogenous bases on inside of molecule
Nucleosome
-Core = 8 histone proteins (+ charged) with DNA molecule (- charged) wrapped twice around (“bead on a string”)
-DNA + histone proteins = chromatin
-Fundamental unit of DNA packaging – allows supercoiling of DNA into chromosomes
-Supercoiling prevents certain genes from being accessed by transcription factors/ enzymes (regulates the process)
Sequences of Nuclear DNA that do or do not code for proteins
- 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 and Chase Experiments Overview
-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
-Note: once a virus infects a cell it “takes over” that cell and forces it to make new viruses
Results of Hershey/ Chase Experiments
-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 (HPPLP)
Hettie Pooped Poppies Longer Please
- Helicase unzips the parental DNA molecule (breaking H-bonds between bases)
Note: in eukaryotes, gyrase and single-strand binding proteins stabilize 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 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)
Semiconservative
DNA Replication process is semiconservative: each daughter molecule produced is half old (parent) strand and half new strand!
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 of Meselson and Stahl Experiments
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
unique, single copy sequences of
DNA) 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
Central Dogma (basic/ fundamental understanding – universal to ALL life) of Molecular Biology:
DNA to RNA to Protein
(Transcription) (Translation)
Transcription Basics
transcribe (writing down a message)= making mRNA (messenger RNA) from DNA
Translation Basics
Translating from one language to another= making a polypeptide chain – a protein - (putting amino acids together) from mRNA
Ribosomes “read” the mRNA code and use it to put amino acids together in the correct order (based on the original DNA sequence) to make a protein
Codon
Set of 3 mRNA base sequences
DNA vs. RNA: Strand
Double vs. Single
DNA vs. RNA: made of
Deoxyribose vs. Ribose
DNA vs. RNA: Bases
Guanine, Cytosine, Adenine, Thymine vs.
Guanine, Cytosine, Adenine, Uracil
Transcription Steps IET + P (In ET and Prince)
- Initiation: (put everything together)
RNA polymerase (IB Student) unwinds DNA strands and binds to promoter on DNA (antisense/ template strand – serves as template for mRNA to be built off of) - Elongation:
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 (A + U and C + G) - Termination: (stops, everything detaches)
RNA polymerase continues until terminator sequence reached (passes in eukaryotes)
mRNA molecule detaches from DNA
RNA polymerase detaches from DNA
-Post-transcription:
In EUKARYOTES: Introns removed to form mature mRNA (only exons remain to be translated into amino acids/ protein) – one gene = many polypeptides (many mRNA from one DNA sequence - alternative RNA splicing by spliceosomes and snRNA’s)
Ribosome Structure
-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 facilitates translation
Contains anticodons (3 bases – complementary to codons on mRNA – will base pair to mRNA during translation)
Amino acid binding site (3’ end at sequence CCA)
Amino acids bound to tRNA using ATP and tRNA activating enzymes (20 of them)
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)
-Genetic Code is universal to ALL life!
-Allows gene transfer between
species (Ex: insulin gene into
bacteria)
Translation Process Steps IET (In ET)
- Initiation: (building the sandwich/ ribosome)
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
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
Nucleosomes and Gene Expression
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 and Gene Expression
-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 and Gene Expression
-Transcription factors – aid in RNA polymerase binding to DNA
-Transcription activators/ transcription repressors
The Environment and Gene Expression
-Can change methylation patterns and/ or affect proteins involved in regulating gene expression/ splicing (wrong genes on or off etc.)
-Chemicals (cigarette smoke, preservatives, pollutants, topical medications/ creams etc.)
Infectious agents (bacteria, viruses, prions)
Proteome
-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 Protein Structure
-Sequence (and number) of amino acids
-Amino acids linked by peptide bonds (formed during translation)
Secondary 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 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 Protein Structure
-Not in all proteins
-Linking several polypeptide chains together (using same bonding as tertiary structure)
-Linking prosthetic group to polypeptide (ex: haem in hemoglobin)
Condensation reactions create…
peptide bonds between amino acids and hydrolysis reactions break peptide bonds between amino acids.
Polar (hydrophilic) Amino Acids (Determined by R groups)
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 (hydrophobic) amino acids (determined by R groups)
form outer portions of membrane proteins (toward phospholipid tails) and proteins embedded in cell membrane; form active sites on enzymes (attract non-polar substrates)
Protein Functions with Examples (shape determines function)
-Enzymes/ catalysts (catalase, amylase, lipase, polymerase etc.)
-Movement (actin, myosin)
-Structure (collagen, elastin, keratin)
-Transport (hemoglobin)
-Defense (immunoglobin, antibodies)
-Hormonal communication (insulin, leteinizing hormone)
Fibrous vs. Globular Proteins: Shape
Long, narrow vs. rounded, spherical
Fibrous vs. Globular Proteins: Amino Acids
Repetitive vs. irregular
Fibrous vs. Globular Proteins: Functions
Structure/ support vs. enzymes/ metabolism
Fibrous vs. Globular Proteins: Solubility
Insoluble in water vs. soluble in water
Fibrous vs. Globular Proteins Examples
Actin, myosin, keratin, collagen vs. Hemoglobin, insulin, amylase
Enzymes are catalysts
they speed up reaction rates, lower activation energy (energy needed to start a reaction), are NOT used up in process
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)
Effect of Temperature on Enzyme Activity
-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 effect on enzyme activity
at optimum enzyme works best/ fastest; below/
above enzymes denature (ions interact with active site/
ionic/ hydrogen bonds)
Substrate concentration on enzyme activity
-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 Metabolic Pathways
(“building” of complex molecules, endergonic – more energy IN to build bonds, often involves condensation, biosynthetic) or
Example: photosynthesis
Catabolic Metabolic Pathways
(“breaking down” of complex molecules, exergonic – more energy released as chemical bonds are broken, often involves hydrolysis, degradative/ digestive)
Example: cellular respiration
Competitive vs. Non-competitive Inhibitors: Shape
Shape is similar to substate vs. shape is not similar to the substrate
Competitive vs. Non-competitive inhibitors: site binding
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
Competitive vs. Non-competitive Inhibitors: Substate Concentration
Increase in substrate concentration reduces inhibition vs.
Increase in substrate concentration does NOT reduce inhibition
Competitive vs. Non-competitive inhibitors: Inhibition
Reversible inhibition vs. Irreversible inhibition (USUALLY)
Lactose Intolerance and Industry
-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)
End Product Inhibition
-The specific steps in a metabolic reaction are called metabolic/ biochemical pathways
-Every metabolic pathway begins with a specific molecule and ends with a specific product
-There are many steps in a metabolic pathway, each of which is catalyzed by its own specific enzyme (because the substrate changes shape after each reaction!)
FINAL product in metabolic pathway can act as allosteric inhibitor
-When end product is present in sufficient amounts, end-product inhibition shuts the “assembly line” down (to prevent the cell from wasting resources)
-When the end-product is present in sufficient amounts it is able to bind to an allosteric site on the first enzyme in the metabolic pathway, causing its active site to change shape, making it non-functional and shutting down the entire pathway.
-A negative feedback mechanism (the outcome of the mechanism causes the opposite effect)– in this case, more product = less activity of the pathway)
–Note: Allosteric regulation can inhibit OR stimulate an enzyme’s activity
Translation Sites
a= awaiting
p= polymerase chain
e= exit
