Lecture 2: Basics

Nucleic acids Side notes: Nucleotides are the building blocks of DNA and RNA Nucleic acids store and transmit genetic information Nucleotides comprise a base, a sugar, and phosphate Nucleotides have additional biological functions, such as energy storage (ATP) and molecular transport Nucleotides contain two types of sugars Two types of sugar molecule are found in nucleotides DNA contains deoxyribose RNA contains ribose, and has an additional oxygen atom In these sugars, the carbons are numbered 1' to 5' (said as "one prime" to "five prime") Nucleotides contain different types of bases DNA and RNA feature two types of base: purines and pyrimidines Adenine and guanine are purines; cytosine, thymine and uracil are pyrimidines DNA contains the bases adenine, guanine, cytosine, and thymine RNA does not have thymine, but uses uracil instead DNA and RNA are polymers of nucleotides Nucleotides are joined by a phosphodiester bond between the 3' hydroxyl of one sugar and the phosphate attached to the 5' hydroxyl of the next sugar NA strands are thus directional - one end has an exposed 3' hydroxyl, the other end has an exposed 5' phosphate By convention, NA sequences are written in the 5' to 3' direction The sugars and phosphates form a repeating unit - the sugar-phosphate backbone The backbone is always the same, but the attached bases vary Top hat l What molecules could catalyze the addition of nucleotides to a sequence? • • • • • Ribosome DNA polymerase Helicase Topoisomerase RNA polymerase DNA synthesis couples favorable and unfavorable reactions Adding nucleotides to DNA requires energy Cells couple that unfavorable reaction with an energy generating reaction Hydrolysis of a nucleotide triphosphate provides nucleotide monophosphate and energy, both needed for the reaction NTPS are hydrolyzed to nucleotide monophosphates and pyrophosphate Pyrophosphate hydrolysis to monophosphates releases additional energy and is coupled to bond formation Complementary base pairs form the DNA double helix Two DNA strands associate via non-covalent hydrogen bonds to form doublestranded DNA Bases pair precisely with their complementary base: A pairs with T with 2 H-bonds C pairs with G with 3 H-bonds These are called Watson-Crick base pairs The sequence of one strand dictates the sequence of the other strand - thus the strands are complementary to one another The two strands are anti parallel - the 5' end of one strand pairs with the 3' end of the other The most energetically favorable formation of double-stranded DNA is for the two strands to wind around one another in a right-handed double helix The hydrophobic bases cluster in the center, away from the aqueous cellular environment Nucleic Acids Side notes: The hydrophilic sugar phosphate backbone interacts favorably with water, and so is on the outside of the molecule Base pairs form a stack on the interior of the helix. Van der Waals interactions between bases stabilize the interactions This "base-stacking" arrangement is very energetically favorable, and is important for the stability of DNA DNA has a major groove and a minor groove A-T and G-C base pairs have similar widths, so the helix diameter is ~20A B-DNA is the predominant configuration. The helix repeats every 10.5 base pairs, and base pairs are 3.4 A apart In B-DNA, the helix forms a major groove (~13 A) and a minor groove (~9 A) The shape and size of these grooves govern interactions with other molecules The sequence of bases in DNA can influence structure: runs of A-T base pairs are more rigid DNA can be in other conformations A-DNA is a right-handed helix, but has 11 base pairs per turn, making the grooves more evenly sized. The A conformation can be induced by DNA binding proteins Z-DNA, a left-handed helix, can result from methylation of cytosine, torsional stress, and high salt concentrations Top hat 2 What biological processes could produce DNA supercoiling? Replication Transcription DNA repair Chromatin Remodeling • All of the above • • • • DNA can be supercoiled To induce supercoiling, a circular molecule is cut and held at one end while the other end is twisted This changes the number of bases per turn When the two ends are stuck back together (ligated) the DNA twists to restore the preferred number of bases per turn This causes the DNA to wrap around itself in a coiled structure Supercoiling can be positive or negative, depending on the direction of the DNA twisting Clockwise winding of the DNA, tending to separate the strands, leads to negative supercoiling Twisting in the counterclockwise direction induces positive supercoiling Linear pieces of DNA can also be supercoiled if one end is immobile Supercoiling is released if one of the DNA strands is cut Supercoiling can be toroidal or intervened RNA is chemically similar to DNA RNA uses uracil instead of thymine RNA has a hydroxyl at the C2 instead of H RNA can base pair with DNA (U base pairs with A) RNA is less stable than DNA The 2' OH in ribose is important for RNA structure and function RNA polymer stability is much lower than that of DNA. DNA is thus more suitable for long term storage and RNA for short term functions The 2' OH facilitates a reaction that can break phosphodiester bonds The 2' OH means that RNA favors an A-type hex rather than the B-type helix of DNA RNA can fold into functional structures Nucleic acids Side notes: Non-coding RNAs often fold into complex, molecule-specific, three-dimensional structures Primary structure is the RNA sequence, in the 5' to 3' direction Secondary structures are short double-helical regions Tertiary structure is the arrangement of the double-helices and single stranded regions in the final configuration of the RNA Increasing numbers of genes are being discovered that produce non-coding RNAs RNA helices are formed from complementary base pairs An RNA double helix is short, usually 6-8 base pairs, in A-helix formation Helices are usually formed with Watson-Crick base pairing, in an antiparallel direction If complementary sequences are close, a hairpin forms (the RNA folds back on itself) In a hairpin, the double-stranded part is the "stem", and the unpaired section is the "loop" In tRNA, three of the double-helices are formed by hairpins, and one is formed from distant complementary regions RNA has base pairing beyond Watson-Crick interactions These non-canonical base pairs often feature chemically modified bases, such as methylation Pairing with modified bases can introduce structural differences such as kinks Another non-canonical pair is G-U, which has two hydrogen bonds (like A-U) This is known as a "wobble" base pair Top hat 3 Are all bases in RNA paired with another base? • Yes • No RNA secondary structure can be identified using phylogenetic analysis RNA sequences can be searched computationally for potential base-pairing regions Because pairing regions are short, this approach can falsely identify interacting regions Phylogenetic analysis (comparison of characteristics between species) helps identify true pairing regions, as these are more likely to be conserved during evolution Differences that occur between species, yet conserve interactions, are known as covariations Amino acids are the building blocks of proteins Amino ands are joined by peptide bonds along a common backbone Amino acid side chains are diverse, with different properties that determine their functions Some are hydrophobic, some hydrophilic A few are amphipathic - having both hydrophobic and hydrophilic characteristics Proteins fold into functional structures A protein folds to a characteristic three-dimensional shape - its conformation Protea folding is driven by non-covalent interactions of the atoms in the polypeptide There are four levels of conformational organization Primary structure is the amino acid sequence Nucleic acids Side notes: Secondary structure refers to localized regions of repeated angular structures such as beta-strands and alpha-helices Tertiary structure refers to the final folded structure of a single polypeptide Quaternary structure refers to the combination of multiple polypeptides in a single protein (note that many proteins only have one polypeptide) Alpha helices are common structural elements in proteins In an alpha helix, the polypeptide backbone forms a right-handed helix Hydrogen bonds form between the C=O of one residue and the n-h four residues away The structure is cylindrical, with side chains protruding round the outside Many helices are amphipathic Hydrophilic residues are on one side of the helix, hydrophobic on the other These often form protein surfaces - the outside interacts with the aqueous cell environment and the inside interacts with the hydrophobic protein interior Beta sheets are common structural elements in proteins Beta streets are formed when peptide backbones (beta strands) hydrogen bond to one another through their carbonyl and amide groups Beta sheets are slightly twisted, largely planar structures Side chains protrude above and below the beta sheet Amphipathic sheets tend to be found at protein surfaces Top hat 4 Do all regions in a protein fold into a specific conformation? • Yes • No Protein folding is driven by hydrophobic interactions Hydrophobic residues associate and exclude water molecules Folding is energetically favorable - entropy increases as water molecules become more disordered Polar residues remain on the outside of the folded protein Internal protein structures are tightly packed, with few gaps Amino acid sequence determines protein structure Proteins can be induced to unfold by changing the conditions (such as heating), and many will then refold when conditions are returned to normal Some proteins (like albumin) cannot refold - these have been denatured Secondary structures, and to some degree tertiary structures, can be predicted from primary sequence Proteins with 50% the same amino acids are likely to have almost identical structures 25% sequence identity yields fairly similar structures Comparing protein sequences can help to identify functional elements The function of a newly identified gene can be predicted based on the amino acid sequence it produces New sequences are compared with sequences of known function Often, similarity of >25% indicates similar fold and similar function Many proteins have key amino acids vital for function Conservation of important amino acids is a strong indicator of similar function Proteins have a modular organization Many polypeptides have more than one domain Nucleic acids Side notes: A domain is a compact region of a polypeptide that can fold on its own Proteins often have multiple domains that fold independently and carry out special functions Proteins can thus be viewed as being comprised of "modules" Macromolecules interact at chemically complementary interfaces Specific interactions between biological molecules govern their functions The strength of molecular interactions comes from the non-covalent interactions formed between them Specificity of interactions and shape of the interaction surfaces affect how strongly molecules interact DNA sequences have district chemical signatures Proteins can interact non-specifically with the negatively-charged DNA backbone Chemical groups are exposed in the major and minor grooves of the DNA helix Each base pair has a characteristic set of groups that are available for interaction DNA-binding proteins most frequently bind in the major groove Alpha helices and two-stranded beta sheets can fit in the major groove Binding in the minor groove introduces a distortion to the DNA that can benefit certain reactions DNA sequences have distinct chemical signatures Different DNA sequences have different combinations of hydrogen bond donors, hydrogen bond acceptors, and methyl groups available DNA-binding proteins form non-covalent interactions with the exposed groups Available chemical groups vary more in the major groove than in the minor groove e.g., g-c and c-g have similar patterns of donors/acceptors, so these are difficult to distinguish by binding proteins Some proteins can recognize particular sequences Interaction specificity between protein and DNA is determined by the DNA sequence and the protein shape & available binding groups. For example: Asparagine can form two hydrogen bonds with an A Polar basic residues are good donors Polar acidic residues are good acceptors An alpha helix in the major groove with side chains forming hydrogen bonds with the DNA