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MolBio - Proteins and DNA Cheat Sheet by

Molecular Biology - Protein Biochemistry, DNA introduction, structure, and replication

Protein Bioche­mistry

Protein Functions
Post-t­ran­sla­tional modifi­cation & Targeting
Different structures reflect unique function
Proteins are made up of amino acids with various side chains
Reversible (addition) or irreve­rsible (removal)
Recogn­ition of specific molecules: hormones, antibo­dies, DNA binding proteins
Amino acids have a hydrogen, central carbon, amino group, side chain, and a carboxyl group
Methyl­ation is adding a CH3 group (eg histones to regulate gene expres­sion)
Movement of molecules: porin, ferritin
Side chains: positive or negative charge, polar or nonpolar, different shapes and sizes
Glycos­ylation is adding sugar molecules (eg cell surface proteins)
Structural functions: components of the cytosk­eleton such as microt­ubules
Primary structure: order of amino acids in a polype­ptide chain, joined by peptide bonds (which are rigid), have a C and N-terminus
Ubiqui­tin­ation is adding a 76 amino acid polype­ptide which denotes protein is ready to be degraded
Enzymes: speed up chemical reactions by lowering the activation energy required
Secondary structure: alpha helix or beta pleated sheet, stabilised by hydrogen bonds
Phosph­ory­lation is adding PO3 group, regulates enzyme function
Tertiary structure: tightly packed 3D structure, noncov­alent intera­ctions between side chains
Targeting is when proteins are transp­orted to where they need to go in a cell
Quaternary structure: complex with 2 or more subunits which can be identical or different
Many proteins have a short signal or locali­sation sequence indicating where they need to go, this is then removed
Many proteins contain several different tightly packed domains, each carries out a specific function

DNA Structure

DNA Structure
Experi­mental Evidence
Chromosome Structure
DNA- Binding Proteins
DNA is made up of nucleo­tides
Chargaff used paper chroma­tog­raphy and looked at base propor­tions. % purine = % pyrimidine
Chromo­somes are long DNA molecules containing genetic inform­ation, have regulatory sequences for proper expression and replic­ation
Proteins bind to specific domains which can have a general affinity for DNA, or are sequence specific
Nucleo­tides have: deoxyr­ibose ring, nitrog­enous base, phosphate group
Wilkins and Franklin used X-Ray crysta­llo­graphy, found DNA is a helix with even structure
Eukaryotic chromo­somes are linear, have a; centro­mere, and telomeres
Transc­rip­tional regulators bind regulatory sequences near promoters to block or stimulate transc­ription (eg lac operon in E.coli)
Purines (adenine, guanine) have 2 rings, pyrimi­dines (cytosine, thymine, uracil) have 1 ring
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
Bacteria have a smaller single circular chromosome
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
DNA is written from 5’ to 3’
Plasmids in prokar­yotes can be passed between cells via conjug­ation
Histones are proteins that DNA wraps around to form chromatin. Not sequence specific
2 H bonds between adenine and thymine, 3 H bonds between cytosine and guanine

DNA as Genetic Material

Chromo­somal Inheri­tance
Transf­orming Principle
Hershe­y-Chase Experiment
Sutton & Boveri invest­igated where genetic material is carried using cytology, and microscopy
Griffith worked on S. pneumo­niae; S strain are pathogenic (have capsule), R strain is not
Bacter­iophage T2 inject genetic material inside E.coli, invest­igated what this material is
Sutton used grassh­oppers, Boveri used Ascaris worms (round­worms). Their chromo­somes are large and few in number, making them easy to observe
When cell extract of dead S strain is injected to mice- no illness. When combined with live R strain and injected- illness
Labelled bacter­iophage with radioa­ctive isotopes. 32P for DNA, 35S for protein to deduce which is genetic material
Discovered chromo­somes are important in reprod­uction and develo­pment
Bacteria are being transf­ormed when combined, hereditary material is being passed
Allowed bacter­iophage to inject unlabelled bacteria. Separated phage from bacteria using blender
Discov­eries matched those of Mendel's, and provided physical basis for his theories
Tested which molecule carries hereditary material, used enzymes which destroy specific molecules.
Centri­fuged. Tested infected bacteria pellet with Geiger counter.
Suggested different combin­ations of chromo­somes could cause variation; discovered genes, and the linear structure of chromo­somes
Discovered DNA is respon­sible for transf­orm­ation. Gene coding for the capsule is passed to R strain from S strain, making them pathogenic
Bacter­iophage labelled 32P had made the bacteria radioa­ctive, indicating DNA is genetic material

DNA Replic­ation

Semi- Conser­vative Replic­ation
Process of Replic­ation
Enzymes for Replic­ation
Leading and Lagging Strands & Telomeres
DNA strands are comple­mentary
DNA strands separate and are used as templates for new strands
Polymerase adds nucleo­tides in a 5’ to 3’ direction, needs primer to start
Leading strand is 5’ to 3’, while the lagging strand is 3’ to 5’ direction
3 theories for replic­ation: conser­vative, semi-c­ons­erv­ative, dispersive
Replic­ation fork- region where DNA is being copied
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)
Replic­ation in lagging strand leads away from fork and is discon­tin­uous. Strand is primed many times, so Okazaki fragments form.
Meselson- Stahl used nitrogen isotopes to test which theory is correct. Grew E.coli in 15N (to make heavy DNA) and transf­erred to 14N
Origin of replic­ation- where the hydrogen bonds are broken and the strands are pulled apart so replic­ation can start
Single stranded binding proteins separate the DNA strands and prevent reanne­aling
Primer removal at the end of Okazaki fragments causes erosion of genetic material, telomeres solve this
Separated heavy and light DNA by ultrac­ent­rif­uga­tion, obtained a liquid gradient.
Humans have multiple origins of replic­ation, E.coli have one
Helicase breaks the hydrogen bonds between bases and unwinds the helix
Telomeres- short stretches of repetitive DNA sequences at the end of chromo­somes, some is lost after replic­ation
Observed using UV light, after 1 generation DNA was hybrid. After 2+ genera­tions it became lighter, proving semi-c­ons­erv­ative replic­ation
Replic­ation is bidire­ctional
Ligase joins the stretches of DNA together into a single strand
Telomeres are effective where DNA needs to be passed on perfectly
Topois­omerase relieves pressure from overwi­nding around the replic­ation bubble by making and resealing breaks in the DNA


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