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Protein Functions
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Structure
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Post-translational modification & Targeting
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Different structures reflect unique function
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Proteins are made up of amino acids with various side chains
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Reversible (addition) or irreversible (removal)
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Recognition of specific molecules: hormones, antibodies, DNA binding proteins
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Amino acids have a hydrogen, central carbon, amino group, side chain, and a carboxyl group
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Methylation is adding a CH3 group (eg histones to regulate gene expression)
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Movement of molecules: porin, ferritin
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Side chains: positive or negative charge, polar or nonpolar, different shapes and sizes
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Glycosylation is adding sugar molecules (eg cell surface proteins)
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Structural functions: components of the cytoskeleton such as microtubules
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Primary structure: order of amino acids in a polypeptide chain, joined by peptide bonds (which are rigid), have a C and N-terminus
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Ubiquitination is adding a 76 amino acid polypeptide which denotes protein is ready to be degraded
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Enzymes: speed up chemical reactions by lowering the activation energy required
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Secondary structure: alpha helix or beta pleated sheet, stabilised by hydrogen bonds
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Phosphorylation is adding PO3 group, regulates enzyme function
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Tertiary structure: tightly packed 3D structure, noncovalent interactions between side chains
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Targeting is when proteins are transported to where they need to go in a cell
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Quaternary structure: complex with 2 or more subunits which can be identical or different
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Many proteins have a short signal or localisation sequence indicating where they need to go, this is then removed
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Many proteins contain several different tightly packed domains, each carries out a specific function
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DNA Structure
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Experimental Evidence
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Chromosome Structure
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DNA- Binding Proteins
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DNA is made up of nucleotides
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Chargaff used paper chromatography and looked at base proportions. % purine = % pyrimidine
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Chromosomes are long DNA molecules containing genetic information, have regulatory sequences for proper expression and replication
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Proteins bind to specific domains which can have a general affinity for DNA, or are sequence specific
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Nucleotides have: deoxyribose ring, nitrogenous base, phosphate group
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Wilkins and Franklin used X-Ray crystallography, found DNA is a helix with even structure
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Eukaryotic chromosomes are linear, have a; centromere, and telomeres
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Transcriptional regulators bind regulatory sequences near promoters to block or stimulate transcription (eg lac operon in E.coli)
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Purines (adenine, guanine) have 2 rings, pyrimidines (cytosine, thymine, uracil) have 1 ring
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Watson and Crick made a model: A-T and G-C hydrogen bonded base pairs, antiparallel strands, right handed double helix, one helical turn every 10.5 base pairs (3.4 nm), major and minor grooves
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Bacteria have a smaller single circular chromosome
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Restriction endonucleases are enzymes that cut DNA at specific sequences. Bacteria use them to restrict virus action, they can be used in the lab to manipulate DNA
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DNA is written from 5’ to 3’
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Plasmids in prokaryotes can be passed between cells via conjugation
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Histones are proteins that DNA wraps around to form chromatin. Not sequence specific
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2 H bonds between adenine and thymine, 3 H bonds between cytosine and guanine
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Chromosomal Inheritance
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Transforming Principle
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Hershey-Chase Experiment
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Sutton & Boveri investigated where genetic material is carried using cytology, and microscopy
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Griffith worked on S. pneumoniae; S strain are pathogenic (have capsule), R strain is not
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Bacteriophage T2 inject genetic material inside E.coli, investigated what this material is
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Sutton used grasshoppers, Boveri used Ascaris worms (roundworms). Their chromosomes are large and few in number, making them easy to observe
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When cell extract of dead S strain is injected to mice- no illness. When combined with live R strain and injected- illness
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Labelled bacteriophage with radioactive isotopes. 32P for DNA, 35S for protein to deduce which is genetic material
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Discovered chromosomes are important in reproduction and development
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Bacteria are being transformed when combined, hereditary material is being passed
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Allowed bacteriophage to inject unlabelled bacteria. Separated phage from bacteria using blender
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Discoveries matched those of Mendel's, and provided physical basis for his theories
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Tested which molecule carries hereditary material, used enzymes which destroy specific molecules.
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Centrifuged. Tested infected bacteria pellet with Geiger counter.
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Suggested different combinations of chromosomes could cause variation; discovered genes, and the linear structure of chromosomes
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Discovered DNA is responsible for transformation. Gene coding for the capsule is passed to R strain from S strain, making them pathogenic
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Bacteriophage labelled 32P had made the bacteria radioactive, indicating DNA is genetic material
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Semi- Conservative Replication
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Process of Replication
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Enzymes for Replication
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Leading and Lagging Strands & Telomeres
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DNA strands are complementary
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DNA strands separate and are used as templates for new strands
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Polymerase adds nucleotides in a 5’ to 3’ direction, needs primer to start
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Leading strand is 5’ to 3’, while the lagging strand is 3’ to 5’ direction
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3 theories for replication: conservative, semi-conservative, dispersive
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Replication fork- region where DNA is being copied
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Primase generates primer (usually RNA), a small stretch of nucleotides in a 5’ to 3’ direction. Removed afterwards and the gap is filled in (by polymerase)
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Replication in lagging strand leads away from fork and is discontinuous. Strand is primed many times, so Okazaki fragments form.
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Meselson- Stahl used nitrogen isotopes to test which theory is correct. Grew E.coli in 15N (to make heavy DNA) and transferred to 14N
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Origin of replication- where the hydrogen bonds are broken and the strands are pulled apart so replication can start
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Single stranded binding proteins separate the DNA strands and prevent reannealing
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Primer removal at the end of Okazaki fragments causes erosion of genetic material, telomeres solve this
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Separated heavy and light DNA by ultracentrifugation, obtained a liquid gradient.
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Humans have multiple origins of replication, E.coli have one
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Helicase breaks the hydrogen bonds between bases and unwinds the helix
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Telomeres- short stretches of repetitive DNA sequences at the end of chromosomes, some is lost after replication
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Observed using UV light, after 1 generation DNA was hybrid. After 2+ generations it became lighter, proving semi-conservative replication
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Replication is bidirectional
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Ligase joins the stretches of DNA together into a single strand
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Telomeres are effective where DNA needs to be passed on perfectly
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Topoisomerase relieves pressure from overwinding around the replication bubble by making and resealing breaks in the DNA
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