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Molecular biology.Kourosh lecture 1- DNA structure.The study of the formation, structure and function of macromolecules essential to life (nucleic acids, proteins), roles in storage and transmission of genetic information.Nucleic acids are linear polymers built of monomer nucleotides. Contain a nitr...

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Molecular biology.

Kourosh lecture 1- DNA structure. The study of the formation, structure and function of macromolecules essential to life (nucleic acids, proteins), roles in storage and transmission of genetic information. Nucleic acids are linear polymers built of monomer nucleotides. Contain a nitrogen ring (organic bases, purine has 2 rings, pyrimidines have 1 ring) linked to a 5 carbon sugar deoxyribose or ribose (pentose) which carries phosphate groups. Phosphate groups are linked by phosphoester bonds.

Nucleoside= sugar and base. Nucleotide= sugar and base and phosphate.

Pentose-phosphate backbone- 5’ phosphate, 3’ hydroxyl (on deoxy/ribose). 5’ to 3’ directionality. Bases are on the same side. There is a negative charge on the outside.

Helix makes a complete turn every 3.4nm, and spaces between the strands form 2 types of binding surfaces- major groove, minor groove. DNA is not planar, it allows for bond angles of carbon atoms. Sugar-phosphate backbone on the outside, bases stacked on the inside. As the percentage of G-C pairs increases, the melting temperature of DNA increases.

Kourosh lecture 2- DNA replication Meselson-Stahl experiment shows DNA replication is semiconservative.

E.coli is grown in N15 and then transferred to N14, samples are removed, DNA is isolated and is analysed by density gradient centrifugation. Both strands serve as templates for replication of a complimentary strand. DNA polymerase is an enzyme that catalyses DNA synthesis. It begins at the replication origin, and uses the old strand as a template, requiring a DNA primer. It adds dNTPs to 3’ OH group. DNA polymerase edits during replication, there are a small number of errors. DNA polymerase only synthesises the 5’ to 3’, so the other lagging strand is made discontinuously as short Okazaki fragments. Fragments are started on RNA primer made by DNA primase and are joined by DNA ligase.

Topoisomerase relieves twists in DNA ahead. They create a momentary break in DNA, so DNA rotates freely and stress is relieved, then the nick is resealed. Also relieves supercoiled DNA. Single strand binding proteins prevent rewinding of DNA. Clamp proteins (PCNA trimer- proliferating cell nuclear antigen) hold polymerase in place, attached to DNA, they work together as a machine and are a huge circular multi-subunit protein. DNA helicase (made of 6 subunits- hexameric ring) is an enzyme that opens up strands of DNA, it hydrolyses ATP and propels it along the strand. dATP stands for 2’ deoxyadenosine 5’ triphosphate. Cellular DNA replication requires primase. DNA polymerase is not essential for RNA replication.

Dr Kourosh- Lecture 3- RNA structure. Genes do not code directly for protein, RNA may be the final product. Half of genes have an unknown function. RNA is made up of ribonucleotide monomers. RNA contains uridine not thymidine. Most RNAs are single stranded (but fold into 3D structures with structural, catalytic and regulatory roles). 80% rRNAs- ribosomal RNAs, component of ribosomes that catalyse protein synthesis. 15% tRNAs- transfer RNAs, decodes mRNA for protein synthesis and brings the correct amino acid to the end of a polypeptide chain. 5% mRNAs- messenger RNAs, codes for proteins.

Regulatory RNAs: miRNA- microRNA- act through RNA interference (RNAi), regulate gene expression (downregulate), single stranded. siRNAs- small interfering RNAs- act through RNA interference, regulate gene expression (turn off/induce), double stranded.

snRNAs- small nuclear RNAs- splices pre-mRNA snoRNAs- small nucleolar RNAs- process and chemically modify rRNA scaRNAs- small cajal RNAs- modify snoRNAs and snRNAs.

Cajal bodies- involved in ribonucloprotein biogenesis, modification of snRNA and snoRNA.

RNA secondary structure. Double stranded loops and hairpins are formed by base pairing. Can be quite stable.

Tertiary structure.

RNA can form unconventional base pairs (G-U). 3D shapes of certain RNAs are stable with specific structural or catalytic roles. RNA can form a double helix, 2’ hydroxyl causes a different helix shape from DNA. Adenine pairs with uracil. In a hairpin stem-loop, the stem is a helix, but the loop has unpaired bases.

rRNA- forms the core of the ribosome, lots of copies in genes. Eukaryotes have 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA. Prokaryotes have 23S rRNA, 16S rRNA, 5S rRNA.

Ribosomes are found free and bound to the rough endoplasmic reticulum. The ribosome is made of 65% rRNA and 35% ribonucleoprotein (RNP). It translates mRNA to a polypeptide chain. In eukaryotes, it is made of 2 subunits- 60S and 40S. In prokaryotes, it is made of 2 subunits- 50S and 30S. Running RNA on (ethidium bromide) gel allows you to see the most abundant rRNA bands, it is used to confirm RNA is intact prior to RT-PCR (reverse transcriptase PCR used to detect RNA expression) or microarray analysis.

tRNA reads the mRNA code and transfers amino acids to a peptide chain.

mRNA is an information carrier and directs protein synthesis. It momentarily associates with ribosomes. It is different in size to reflect the difference in protein size. It is made and then rapidly broken down. The secondary structure is not very stable- unfolds for ribosome and tRNA. It is not chemically modified. It goes through processing between transcription and translation.

Dr Kourosh- lecture 4- RNA transcription. RNA is synthesized 5’ to 3’. RNA polymerase catalyses phosphodiester bond formation. rNTP is specified by DNA template strand and is added to the 3’ hydroxyl end of the growing RNA strand. Pryrophosphate is released. RNA polymerase begins at the transcription start site (+1) moving downstream. The promoter region is upstream of the start site. The DNA template strand is transcribed and is complimentary (identical to non-template strand but with U instead of T). DNA is transcribed by RNA polymerase. Nucleotidyl transferase catalyses the linkage of ribonucleotides and does not need a primer. It also proofreads. RNA polymerase I- transcribes 45S rRNA precursor to 5.8S, 18S and 28S rRNA. RNA polymerase II- transcribes precursor mRNAs, miRNA, siRNA, snoRNA and most snRNA. It is made of 10 different protein core subunits. It has a pore for rNTP entry and an exit groove for RNA. It has an active site with a heteroduplex of DNA and RNA. RNA polymerase III- transcribes tRNAs, 5S rRNA and other small RNAs. Initiation- RNA polymerase finds and binds to the gene promoter, using transcription factors. The DNA strands are melted around the start site (transcription bubble). The template strand enters the active site and phosphodiester bonds between rNTPs are formed.

Elongation and termination- RNA polymerase dissociates from the promoter and transcription factors, and moves along the template strand. The elongation complex is very stable. The DNA sequences signal termination and the primary transcript is released, so polymerase dissociates.

Initiation in eukaryotes requires TBP from TFIID (TATA binding protein) that recognises the TATA box, TFIIH which uses ATP to open up DNA and phosphorylate C terminal of polymerase. The phosphorylation changes polymerases conformation and releases it from other transcription factors, so initiation can begin.

TATA box is a cis-regulatory element (stretch of DNA where TFs can bind). The AT sequence facilitates unwinding. Eukaryotes have a Hogness box. Prokaryotes have a Pribnow box.

Transcriptional activators are gene-specific regulatory proteins which bind to DNA sequences and attract RNA polymerase. Mediator protein complex mediates communication between transcriptional activators and polymerase. Chromatin remodelling proteins are also needed.

Lots of protein subunits are needed for initiation, and order of assembly and transcriptional activators is gene specific. Polymerase is released from this complex for elongation.

RNA processing- 5’ capping with a 7-methylguanosine cap, which protects degradation, signifies the 5’ end of mRNA and helps with export and translation. 3’ tail polyadenylation signifies the 3’ end of mRNA and helps with export and translation. Introns are spliced, exons are ligated and expressed. The 5’ and 3’ untranslated regions are kept during processing but are not translated, they are regulatory. Introns are spliced out, dictated by consensus sequences. Pre-mRNA splicing- 2 sequential phosphoryl-transfer reactions (transesterifications), joins exons and removes the intron as lariat. It is carried out by 5 snRNAs (U1, U2, U4, U5, U6). The spliceosome recognises the 5’ splice junction, the branch-point site and the 3’ splice junction. RNA rearrangements require ATP hydrolysis. Cellular RNA synthesis is also known as transcription. Mature eukaryotic mRNA has a poly(A) tail.

The genetic code is degenerate. RNA splicing occurs using the ribozyme.

Dr Kourosh- lecture 5- protein biosynthesis. RNA splicing- branch point binding protein (BBP) and U2AF recognise the branch point. snRNPs mediate RNA rearrangement. Adenine at the branch point attacks the 5’ splice site. Lariat is formed and exons are joined. ATP is needed for splicing, but it is worth it because- it favours emergence of new proteins, most have common protein domains, it increases the coding potential of the genome as multiple proteins can be created. The genetic code has degeneracy at the third position. AUG is the start codon (methionine) and dictates the reading frame. Frameshift mutations are insertions or deletions of nucleotides that disrupt the reading frame and do not normally generate a functional protein. Some amino acids have more than one tRNA. The third position may be a wobble position that allows unconventional base pairing.

Translation Activation- Aminoacyl-tRNA synthetase pairs the right amino acid to the tRNA, using a high energy ester bond. ATP is hydrolysed. They have separate recognition sites for amino acids and tRNAs. Hydrolytic editing is performed in case of errors. The amino acid is attached to a conserved CCA sequence at the 3’ end. Initiation- assembly of the ribosome subunits and mRNA. Elongation- amino acids added to the polypeptide chain. Termination- release of polypeptide chain from tRNA and dissociation of ribosomes.

Amino acids are activated by the addition of AMP. The adenylated intermediate is transferred to the hydroxyl group on tRNA.

Dr Kourosh- lecture 6- protein biosynthesis 2.

Initiation- begins with initiator tRNA, eukaryotic initiation factors and the small ribosome subunit. Then the small subunit recognises and binds mRNA through elF4. The complex moves 5’ to 3’ and scans for AUG. Leaky scanning can produce more than 2 proteins. ATP dependent helicases open up RNA secondary structure. Initiation factors dissociate, and the large subunit binds. Aminoacyl-tRNA binds to the A site and the first peptide bond forms.

Bacterial mRNA- often polycistronic- encodes several different proteins from the same mRNA. The ribosome is often in the middle of the mRNA. Has a Shine-Dalgarno sequence- ribosome binding site.

Elongation- incoming aminoacyl-tRNAs are linked to EF1a-GTP. Base pairing at anticodon hydrolyses GTP, there is a ribosome conformational change and tight binding of aminoacyl-tRNA, then EF1a-GTP is released. The ribosome conformation facilitates the peptide bond formation (peptidyl transferase activity by the large rRNA). The ribosome translocates one codon- empty tRNA moves to exit site so the A site is open and elongation begins again.

Peptide bond formation- condensation reaction between carboxyl of one amino acid with amine of another amino acid, water is released.

Termination- stop codons are UAA, UAG and UGA. Eukaryote release factors- eFR1 looks like tRNA and binds the stop codon. It promotes cleavage of peptidyl-tRNA. Other eRFs promote dissociation of the ribosome and mRNA. Translation efficiency is increased by polyribosomes. Multiple ribosomes simultaneously translate a single mRNA. It has a circular structure. Ribosomes are rapidly recycled, helped by elF4E and elF4G. Multiple initiation allows more protein to be translated, they can build the same mRNA sequentially.

Proteins assume their 3D shape when exiting the ribosome. They are helped by heat shock proteins (molecular chaperones). DNA and RNA polymerase are different structurally, in requiring a primer, in error rate and proof reading. Transcriptional initiation requires- transcriptional activators, chromatin remodelling proteins and tRNA. RNA processing is carried out in eukaryotes only.

Dr Kourosh- lecture 7- protein structure to function. Function of proteins is regulation, signalling, structural, enzymes, movement, transport.

Primary- linear sequence of amino acids with peptide bonds. The structure is very strong, and requires high energy input to break the bonds (acids, peptidases, proteases). The peptide bond is planar and allows rotation around the a carbon. Side chains determine the chemical properties. Secondary- folding of polypeptide chain into a helices or b sheets. It is non-random and partly determined by the polarity of side chains, sometimes assisted by folding enzymes, and stabilized by intra-chain noncovalent bonds. A helix is formed from hydrogen bonding between every 4 th peptide bond. A rigid cylinder is formed. The hydrogen bonds are formed between groups of the backbone, not the side chain. There is a complete turn of the spiral every 3.6 residues, and they are common in transmembrane proteins with non polar side chains facing out. In beta sheets- there are strands of amino acids packed together laterally, and hydrogen bonding occurs between adjacent strands. R groups stick out above and below the sheet. The fixed bond angles produce pleating, and strands are parallel or antiparallel. Tertiary- 3D structure of polypeptide. Quaternary- multiple polypeptides.

Water makes proteins fold so there is a hydrophobic core and polar side chains are on the outside and can form hydrogen bonds with the water.

Hydrophobic amino acids (side chain buried inside the protein core) are non-polar. Hydrophilic amino acids are polar and can participate in hydrogen bonding.

Multiple weak bonds combine (hydrogen bonds, electrostatic interactions, van der waals attractions) to form a stable shape.

Combinations of secondary structures contribute to the overall structure of the protein= supersecondary structure/protein folds/protein motifs. Coiled-coil motif- 2, 3 or 4 a helices wrapped around each other. They are often leucine zippers (transcription factor), or seen in fibrous proteins like tropomyosin, and HIV entry protein.

Protein domains are distinct regions of the tertiary structure. Lots of amino acids fold independently. It is a modular unit of which large proteins are built. Functional domains exhibit the activity characteristic of the protein (catalytic kinase domain, ATP binding domain, DNA binding domain). Topological domains are defined spatially- transmembrane domain, cytoplasmic domain, extracellular domain.

Proteins evolve new functions by joining domains in new combinations. Globular domain- compact 3D structure from a mix of a helix and beta sheets (more soluble) is seen in proteins with catalytic, regulatory and signalling functions. (enzymes) Fibrous domain- elongated insoluble filaments (mainly beta sheets) is seen in proteins with structural and contractile functions. (keratin, elastin, collagen) Glycoprotein on influenza virus- hemagglutinin- is a combination of globular and fibrous (HA1, HA2) protein chains, and has 3 identical subunits of both of them together. It is stabilized by lateral interactions in the globular domain.

Macromolecular machine- DNA replication, RNA transcription, RNA splicing. Ligands are ions, small molecules, macromolecules that form initial weak bonds at a specific binding site and causes a conformational change.

Jose- lecture 8- PCR and cloning Gene- basic physical and functional unit of heredity, made up of DNA and act as instructions to make proteins. Genome- organisms complete set of DNA, with genes, non coding regions and DNA or organelles. Genotype- collection of an individual’s genes. Phenotype- observable traits of an individual. Gene expression- transcribing a gene to produce mRNA (translation will follow).

Gene cloning- transferring DNA from one organism to another, to study a gene and see how it is expressed in a different context.

Genes cloned in E.coli- GFP originally from fluorescent jellyfish. Used to study gene expression and protein location inside a cell. Can have RFP, YFP, CFP.

DNA cloning: Get enough DNA from PCR. (Use template DNA, DNA primers, dNTPs, thermophilic DNA polymerase with a Mg2+ buffer). Denaturation at 95, annealing at 55, elongation at 72. DNA strands duplicate after each cycle.

Digest with restriction endonucleases. Ligate the DNA into a vector. Transform into E.coli. Select the transformants.

DNA fragments can be separated using gel electrophoresis. DNA molecules have a negative charge and can be separated according to size and loading them into an agarose gel, and applying an electric current. Size is determined by the distance travelled, compared against a known standard ladder. PCR can be used to insert engineered sequences into the DNA template. New DNA short motifs are included at the beginning of the primers in the reaction. Only the few initial template sequences will not have the modification, but the rest of the cloned DNA from PCR will. The Sanger method can also be used to sequence DNA- it uses fluorochromes and produces a chromatograph. PCR can detect a species of a pathogen by designing a primer to anneal to a gene in the target species. If the PCR works, it has annealed and the target DNA was present there. PCR can be used in forensics for DNA fingerprinting to identify people, paternity tests, archeology, anthropology. Primers are designed to anneal to repetitive sequences in the genome that vary between individuals.

Restriction endonucleases are synthesized by certain microorganisms. They cut DNA internally at specific sites. They defend against foreign DNA and have their own DNA protected by methylation. They are named from the organism they came from. Type II enzymes are the most useful. They recognises palindromes which are specific sequences, and can leave overhanging sticky ends (staggered cut) or blunt ends (direct cut) where ligation is less efficient as it does not have protruding strands and cannot anneal.

Ligase enzymes join DNA molecules together- a 5’ phosphate group to a 3’ OH group to form a phosphodiester bond. Involved in DNA repair, lagging strand synthesis (okazaki fragments), rejoining DNA after unravelling during replication, integrating foreign DNA.

All ends can ligate with ends produced by the same enzyme. Sticky ends will ligate with another sticky end produced by another enzyme if it is a compatible end. A blunt end will ligate with any other blunt end- all blunt end enzymes are compatible. Blunt end- blunt end Sticky end- sticky end, if compatible Blunt end- sticky end, if compatible

Plasmid vectors are small circular molecules of double stranded DNA. They replicate independently of the chromosome and are not essential for bacterial growth, but can provide a selective advantage under certain conditions (antibiotic resistance). They are used in molecular biology to introduce and grow foreign DNA into bacteria. Functions- some contain genes for the conjugation pilus in bacterial mating, some contain genes coding for toxins for pathogenicity. Plasmid structure- has an origin of replication, a selectable marker (antibiotic resistance), a multiple cloning site (polylinker), a marker for recombinants. May have an inducible promoter, ribosome binding sites and termination sequences so the gene can be expressed. A...