Nucleic Acids - Lecture notes 1 PDF

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Summary

Nucleic AcidsFeatures Macromolecules that store and express genetic information  2 main types – DNA and RNA (both as a carrier of instructions)Why study nucleic acids? Genes are made from nuclei acids and determine our characteristics both as a species and an individual  Genes determine characte...

Description

Nucleic Acids Features  

Macromolecules that store and express genetic information 2 main types – DNA and RNA (both as a carrier of instructions)

Why study nucleic acids?    

Genes are made from nuclei acids and determine our characteristics both as a species and an individual Genes determine characteristics between individuals in species e.g. blood groups Faulty genes cause diseases (20%-30% of childhood deaths) DNA sequencing and genetic manipulation is important in research, medicine and biotechnology

The Central Dogma    

Shows the flow of genetic information through a biological system DNA is replicated – very stable however useless on its own (needs to be copied into a more usable molecule) Transcribed into RNA as it more usable – disposable form and can sometimes be copied back into DNA Translated into proteins – going from 4 amino acids into over 20 amino acids

Structure   

DNA and RNA are composed of subunits called nucleotides Each nucleotide has 4 subunits Each nucleotide contains: - 5 carbon sugar - Nitrogenous base - Phosphate group

Sugar  

Deoxyribose in DNA No hydroxyl group on C2

Bases       

Adenine Thymine Guanine Cytosine Uracil (replaces thymine in RNA) Adenine and Guanine are purines (2 rings) Thymine, Cytosine and Uracil are pyrimidines (1 ring)

Nucleoside

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A nucleoside is formed when a base is linked to the C1 of a deoxyribose molecule Deoxyribose + Adenine = Deoxyadenosine ” + cytosine = Deoxycytidine ” + guanine = Deoxyguanosine ” + thymine = Deoxythymidine

Nucleotide   

Nucleoside with 1 or more phosphate group attached to C5 When naming a nucleotide take the name of the nucleoside and add “monophosphate” Deoxyadenosine monophosphate (dAMP) Deoxycytidine " (dCMP) Deoxyguanosine " (dGMP) Deoxythymidine " (dTMP)

Phosphodiester Bond     

Nucleotides are linked by phosphodiester bon that run from 5’-3’ 2 ester links with a phosphorus atom in the middle Nucleic acids are negatively charged (caused by phosphate being a proton donor) Nucleic acids have a distinct 5’ to 3’ polarity Sequence can be written down according to bases from 5’ to 3’ end

Double stranded  

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Some viruses contain single stranded DNA but in all prokaryotes and Eukaryotes DNA is double stranded Double stranded DNA has 2 complementary DNA strands, running in opposite directions and held together by hydrogen bonds between bases with a sugar-phosphate backbone A-T (2 hydrogen bonds) C-G (3 hydrogen bonds) Base pairing compromises of 1 purine and 1 pyrimidine Base pairs are the same size Base cannot bond with any other base: - If C and T were to bind together the base pair would be narrower as they are both pyrimidines (1 ring) so DNA molecule would constrict inwards - If A and G were to bind together the base pair would be wider as they are both purines (2 ring) so DNA molecule would bulge outwards A base pair always contains one purine and one pyrimidine Will always have the same proportion of (A+T) and (G+C) – Chargaff’s rule

Double helix  

Double helix secondary structure Stands run antiparallel with each other

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Right handed helix Sugar and phosphates are on the outside Bases bind the 2 strands across the middle (hydrophobic area between base pairs) Base interact with bases above and below by hydrophobic interactions and Van de Waals – helps to stabilise the DNA molecule Diameter of DNA = 2nm Height of DNA = 3.4nm per turn 10 base pair per turn Has major and minor groves DNA has the same structure no matter where it comes from, the sequence differentiates it

Major and Minor Grove   

Major grove – where the sugar phosphate strands are far apart and basses are relatively accessible Minor grove – where the sugar phosphate strands are close together and the bases are relatively inaccessible DNA sixe is usually described in base pairs

Alternative DNA Conformations    

DNA is a flexible structure due to free rotation around phosphodiester bond and glyosidic bond B-DNA – The common form, DNA is B-DNA, most or all cellular A-DNA (or double-stranded RNA) – 11 bp/turn, slanted base-pairs, major groups are deeper and minor groups are shallower (forms when DNA is dehydrated) Z-DNA – 12 bp/turn, left-handed, Zig-zag backbone (may form if DNA contains long runs of alternating G and C (e.g. CGCGCG))

Supercoiling       

If DNA is under wound or overwound it will become supercoiled - the molecule twists around itself Under winding generates negative supercoils – advantageous for DNA transcription as it is easier to unwind local regions Over winding generates positive supercoils Unwinding DNA is an important biological process Unwinding a DNA molecule with fixed ends introduces positive supercoils Pulling both ends apart forms Topoisomerase are enzymes that are introduced in order to remove supercoils

Denaturing of DNA 

Loss of non-covalent bonds – van de Vaals, H bonds

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When heated at 70 - 110°C (or exposed to alkaline conditions) DNA becomes denatured The strands separate If allowed to cool slowly the strands will re-anneal If cooled quickly the strands will not have sufficient KE in order to re-anneal Denaturation can be monitored by measuring absorbance of UV light at a wavelength of 260 nm Single stranded DNA absorbs more UV than double stranded DNA – Therefore UV absorbance rises as DNA denatures (the “hyperchromic shift”) Melting temperature (Tm): The temperature needed to denature 50% of the DNA in a sample. Tm is increased in: - DNA with high content of GC base-pairs, since there are more H-bonds between the strands. - The presence of cations (e.g. Na+) – these reduce repulsion between negatively charged phosphate groups on the two strands

How Does RNA Differ to DNA? 

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There are three key structural differences between DNA and RNA: - RNA has ribose instead of deoxyribose - The OH on the 2ʹ carbon makes RNA denser, and more reactive (less stable) than DNA Contains adenine, cytosine and guanine (like DNA) but has Uracil (U) instead of thymine. Like thymine, uracil is a pyrimidine that can form a base pair with adenine RNA, like DNA, consists of nucleotides linked by phosphodiester bonds However DNA is usually double stranded and RNA is usually single stranded Double stranded RNA resembles A-DNA rather than B-DNA. RNA molecules may fold back on themselves to form complex secondary structures, e.g. transfer RNA (tRNA). The greater structural flexibility of RNA allows a greater number of function: - Carry information (messenger RNA) - Act as a transporter (transfer RNA) - Act structurally or catalytically (e.g. ribosomal RNA)

Proof That Genes Are Made Of DNA  

Alterations in DNA cause genetic diseases Introduction of foreign DNA into an organism may alter its characteristics

Bacterial Genomes

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Most often prokaryotes have only one chromosome that is circular (no free ends). An E. coli cell is only 2 mm long but contains 1.3 mm DNA DNA needs to be compressed by binding to protein to form the nucleoid (but not enclosed by a membrane, i.e. no nucleus) Contains loops of supercoiled DNA and proteins Most prokaryote DNA is organised into genes and encodes protein (i.e. coding DNA)

Plasmids        

In addition to a chromosome, prokaryotes may contain plasmids. Accessory circular DNA molecules separate from the chromosome Readily passed from cell to cell Can be exploited in genetic manipulation Plasmids carry non-essential genes, e.g. for antibiotic resistance A cell may contain between 1 and 200 copies of a particular plasmid size range 1kb - 400kb Have an origin of replication

Eukaryotic Genomes 

Eukaryotic cells have DNA in nucleus, mitochondria and (in plants) chloroplasts

Mitochondrial Genome   

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A circular DNA molecule, about 16,500bp in length. Multiple copies present in each mitochondrion 37 genes: 22 encoding tRNA - 2 encoding rRNA - 13 encoding mRNA Almost all mitochondrial DNA is organised into genes Most mitochondrial proteins are encoded by genes in the nucleus Almost no non-coding DNA in the mitochondrial genome

Eukaryotic Nuclear Genome  

Most eukaryote DNA is not organised as genes and does not encode protein (non-coding DNA). Approx. 1.2 % of human DNA encodes protein About 50% of the human genome is repetitive DNA i.e. non-coding sequences present in thousands of copies

Eukaryotic Chromosomes

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Eukaryotes have a number of linear chromosomes (from 1 pair to over 200) that are only visible at mitosis/meiosis Humans - 46 chromosomes - 23 pairs, one of each pair from each parent. The two copies of a particular chromosome are homologous Chromosomes are present but not visible during interphase Mitotic chromosomes are two identical chromosomes (sister chromatids) joined together at the centromere

Histone Proteins 

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There are five types of histone protein in eukaryotes: - Histone H1, - Histone H2A, - Histone H2B, - Histone H3 - Histone H4 All histones are basic – approximately 20% of amino acids are arginine or lysine. Highly conserved between species Basic side chains are positively charged at neutral pH – good at forming electrostatic interactions with negatively charged DNA

Nucleosomes 

When interphase nuclei are lysed and their contents viewed though the electron microscope we see beads on a string”, structure is sometimes known as the 10 nm filament - The strings are DNA - The beads are nucleosomes

Nucleosome Structure 

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Nucleosome is 146 bp DNA wrapped around “core particle” containing: - 2 molecules of histone H2A - 2 molecules of histone H2B - 2 molecules of histone H3 - 2 molecules of histone H4 Nucleosomes can be thought of as the subunits of chromosomes and chromatin

Histone H1  

H1 Binds to DNA outside core particle sealing DNA to particle Seals the DNA to the core particle

Higher Order Structures  

Long, regular structures made from nucleosome (e.g. a helix with 6 nucleosomes per turn) probably do not exist in vivo (inside cells) Instead, nucleosomes appear to be packed into the nucleus of interphase cells in disordered chains with a varying density of nucleosomes

The Mitotic Chromosome Has a Scaffold of Non Histone Protein

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The scaffold contains condesins and topoisomerases that anchor loops of nucleosomes

Eukaryotic Genes can be regulated by Modification of Histone Proteins     

For example, acetylation of lysine residues Acetylation removes positive charge from side-chain and so weakens interaction between histones and negatively-charged DNA – addition of OCH3 group DNA associated with acetylated histones is much more readily transcribed than DNA associated with unmodified histones – easier for enzymes to get DNA into is active site Histone acetylases can turn genes on by reducing the attraction between DNA and protein Histone deacetylases can switch genes off causing the DNA to enter a repressed inactive state

Eukaryotic Vs Prokaryotic Genomes

Structure of Eukaryotic Chromosomes during Mitosis (Mitotic Chromosomes)   

Packing ration = Length of DNA/Length of structure DNA is packed into smallest human chromosome (during mitosis) is 2 m long but contains 14 mm (14,000 m) DNA Therefore 14,000/2 = 7,000

What are Eukaryotic Chromosomes Made of?   

1/3 DNA (one long molecule) 1/3 histone proteins – group of 5 proteins 1/3 non-histone proteins

DNA Replication is Semi Conservative  

1 of the 2 parent strands are conserved in each of the new DNA molecules The strands first unwind and each acts as a template strand

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Each daughter molecule has one new strand and one pre-existing strand of DNA, i.e. semiconservative Unwinding occurs a few hundred nucleotides at a time

DNA Polymerase       

E coli have 5 types of DNA polymerase (1,2,3,4,5) DNA polymerase 1+3 are involved with DNA replication All four deoxynucleoside triphosphate e.g. dATP, dCTP, dGTP, dTTP A region of single stranded DNA (template is required for DNA polymerase to act upon) A primer (short piece of nuclei acids that is based paired to the template molecule) acts at a start point, Primer must have a 3’-OH group DNA is synthesised in the 5’-3’ direction in relation to the primer

DNA Polymerase Reaction    

Oxygen from (OH) group acts as a nucleophile – forms a new bond with inner most P atom High energy phosphate bond between 1st and 2nd phosphate on pppC pC is added to the chain pp is left over and phosphate bond breaks to form 2Pi

Other Enzymatic Activities of DNA Polymerase I 1.  5’ to 3’ exonuclease – Binds to the end of the strand and degrade DAN from the 5’ end  5’ to 3’ exonuclease and DNA polymerase can combine activity can combine to allow for Nick translation  Exonuclease bind to the 5’ end and begins degradation  DNA polymerase bind to the 3’ and begins synthesis simultaneously 2.  3’ to 5’ exonuclease – allows polymerase 1 and 3 to remove incorrect nucleotides from newly made DNA (Proof read – removes errors)  Primary is not bonded to a base pairs that is bonded to the template  DNA replication stops  3’ to 5’ exonuclease removes incorrect nucleotide  DNA replication can continue Comparison of DNA Polymerase I and III  

DNA polymerase III is the main enzyme involved in DNA replication Possessive enzyme – stays attached to its template repeating the reaction without falling off and have to re-associate

Replication Fork   

One strand runs 5’ – 3’ The other runs 3’ – 5’ Leading strand runs 5’ – 3’

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Lagging strand runs 3’ – 5’ (wrong direction)

DNA Replication in E.coli 1. Partial unwinding of DNA by helicase – uses to ATP molecules for each base air it is going to split (2 ATP molecules for each base pair) - Unwinding causes tighter coiling to compensate for the unwinding at a particular region - DNA gyrase indroduces negative supercoiling in order to counteract positive spercoiling 2. Single stranded binding protein binds to DNA to prevent the DNA strands from re-associating with each other (binds 1 every 10 nucleotides) - Removed by DNA polymerase 3. Primers are formed from RNA (made by an enzyme called primase) – Binds to the replication fork to synthesis primer 4. DNA synthesis by DNA polymerase III – Synthesis of Leading strands heads towards the fork 5. Progressive unwinding – DNA polymerase only work in 5’ – 3’ direction 6. Lagging strand has a new RNA primer attached (discontinuous – appear to grow in the wrong direction) - Okasaki fragments are around 1000 base pairs in length - Leading strand has one primer that replicated DNA continuously 7. Removal of RNA primers by Nick translation by DNA polymerase I 8. DNA ligase – seals the gap by forming phosphodiester bond In All Species     

DNA is semiconservative Bidirectional Semi discontinuous Dependant on RNA primers DNA Replication in Bacteria and Eukaryotes

Transcription 

Either strand can be used as the coding strand

Products of Transcription   

Genes are located in the nucleus, but protein synthesis occurs in the cytoplasm, so need an intermediate to carry information from nucleus to cytoplasm. This intermediate is messenger RNA (mRNA). Transcription also generates other RNAs, e.g. transfer RNA (tRNA) and ribosomal RNA (rRNA).

What Does RNA Polymerase Require for Activity?    

Uses the same mechanism as DNA polymerase All four nucleoside triphosphates, i.e. ATP, CTP, GTP, UTP A template DNA molecule A promoter (but NOT a primer)

Transcription in E.Coli  RNA polymerase must recognise the start of the gene – the DNA sequence of the promotor  Bind to the promoter and then copy DNA of gene into RNA 1. Initiation - RNA polymerase binds to the promoter. - DNA strands partially unwind. - RNA synthesis begins - Carried out by holoenzyme form of RNA polymerase – a hexamer consisting of subunit structure α2ββ’ωσ (can recognise specific sequences) 2. Elongation - RNA polymerase moves along the DNA molecule synthesising an RNA copy. - Only 15-17bp of DNA are unwound at any one time - Carried out by core enzyme – pentamer with sub unit structure α2ββ’ω (cant recognise specific sequences) 3. Termination - RNA polymerase dissociates from DNA releasing the new RNA molecule Summary   

Holoenzyme binds to the promoter The σ subunit then dissociates leaving the core enzyme which carries out elongation After termination the core enzyme dissociates from the DNA and binds to another σ subunit to reform the holoenzyme

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Promoters used by RNA polymerase II usually consist of a core promoter that may include the pribnow box and -35 hexamer (Consensus sequences)

Transcription in Eukaryotes 

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Differs from transcription in bacteria in that: -

Eukaryotes have separate RNA polymerases for mRNA, rRNA and tRNA.

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Eukaryotes make a 1º transcript (aka pre-mRNA) thatis processed in the nucleus to form mRNA.

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Eukaryotic promoters differ from those in bacteria.

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Eukaryotic RNA polymerases cannot directly recognise the promoter

Eukaryotes have 3 types of RNA polymerase – Prokaryotes have 1 type -

1 (rRNA)

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2 (mRNA)

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3 (tRNA)

Genes have a different arrangement in eukaryotes (exons and introns)

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And one more proximal promoter elements (aka upstream promoter elements – UPE’s) Efficient transcription of eukaryotes genes also require more distant sequences called enhancers Enhances increases the efficiency with which a gene works/ increases transcription rate Promoters used by RNA polymerase II require accessor proteins called transcription factors The core promoter interacts with general transcription factors – where RNA polymerase will bind (TATAAA) box Proximal promoter are sequences that are protein binding sites – help with gene regulation (determines whether the gene is switched on or off) RNA polymerase II copies both exons and introns – primary transcription At the start of the RNA a cap is added End is cleaved and poly A tail – all mRNA will end with around 250 A residues RNA splicing then occurs – breakage of bonds between exons and introns

The Role of Proximal Promoter Elements     

Promoters are DNA sequences located in the 5' region adjacent to the transcriptional start site RNA polymerase and accessory proteins (transcription factors) bind to the promoter to initiate production of an mRNA transcript Eukaryotic genes will not normally be transcribed unless addition transcription factors re also bound to proximal promoter elements These transcription factors stimulate formation of the pre-initiation complex and may also recruit histone acetylates Pre-initiation complex is a large complex of protein that assemble at the promoter and bind to RNA polymerase

Translation Ribosomes         

Provides and interface where mRNA and tRNA can come together Has 3 binding sites Catalyses the formation of peptide bonds Composed of protein and rRNA )2:1 ratio to protein) Size is measured in (S) – sedimentation rate du...