Nucleic Acid Structure 2024 PDF

Nucleic acid structures By the end of this lecture, you should be able to: Describe the structure of DNA Describe how RNA differs from DNA Describe the different types of RNA and relate structure to function  Sugar-phosphate backbone 5′ end...

Nucleic acid structures By the end of this lecture, you should be able to: Describe the structure of DNA Describe how RNA differs from DNA Describe the different types of RNA and relate structure to function  Sugar-phosphate backbone 5′ end Nitrogenous bases Pyrimidines 5′C 3′C Nucleoside Nitrogenous base Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA) 5′C Purines 1′C Phosphate 3′C group Sugar 5′C (pentose) Adenine (A) Guanine (G) 3′C (b) Nucleotide Sugars 3′ end (a) Polynucleotide, or nucleic acid Deoxyribose (in DNA) Ribose (in RNA) (c) Nucleoside components  X-ray diffraction Crystals of a molecule are bombarded with a beam of X-rays. The rays are bent, or diffracted, by the molecules they encounter, and the diffraction pattern is recorded on film When analysed mathematically, the diffraction pattern can yield information about the three-dimensional structure of a molecule.  Rosalind Franklin Performed X-ray diffraction studies to identify the 3-D structure of DNA using Maurice Wilkins’ DNA fibres, contributing to the discovery of the structure of DNA (a) Jewish Chronicle Archive/Heritage Image Partnership Ltd/Alamy Stock Photo; (b) Omikron/Science Source Access the text alternative for slide images. © McGraw Hill, LLC 5  Rosalind Franklin – an X ray crystallographer imaged two forms of DNA The A form is a crystalline form – more detailed images. B is ‘wetter’ – simpler image. X shape is diffraction signature of a helix  X ray diffraction– Photo 51 Franklin and Wilkin's deduced that there was one nucleotide every 3.4 Å (0.34 nm), that there were 10 nucleotides/turn of the helix and that the helix was 20 Å wide. Each line, shows a ‘rung’ on the DNA – and each full twist has 10 nucleotide units The basic parameters of the DNA molecule provided by ‘Picture 51’ provided important information for Watson and Crick. The helix was 20 Å wide. Watson and Crick’s modelling showed:  Chargaff’s Rules Erwin Chargaff determined that: Always an equal proportion of two-ringed purines (A and G) and single-ringed pyrimidines (C and T) Amount of adenine = amount of thymine Amount of cytosine = amount of guanine The ratio of A-T and G-C varies by species © McGraw Hill, LLC 9 The Watson-Crick DNA double helix model  James Watson and Francis Crick – 1953 Deduced the structure of DNA using evidence from Chargaff, Franklin, and others Did not perform a single experiment themselves related to DNA Key insight of their model was each DNA molecule was made of two intertwined chains of nucleotides, that is a double helix structure For which they won the Noble prize along with Maurice Wilkins © McGraw Hill, LLC 11  The polynucleotide strands held together by H bonding between base pairs A+T 2 H-bonds Purine-pyrimidine pairs G+C 3 H-bonds Stacking of nucleotide bases In a nucleotide the base is hydrophobic inside and there are two very hydrophilic groups: a negatively charged phosphate and a sugar (carbohydrate) group. The two very hydrophilic groups form H- bonds and will interact strongly with water. The hydrophobic surfaces of the bases are found in the centre of the DNA molecule, whereas the sugars and phosphates at the periphery, in contact with water. The stacking arrangement accomplishes two things: It makes the molecule both compact and very strong.  Sugar-phosphate backbone 5′ end Nitrogenous bases Pyrimidines 5′C 3′C Nucleoside Nitrogenous base Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA) 5′C Purines 1′C Phosphate 3′C group Sugar 5′C (pentose) Adenine (A) Guanine (G) 3′C (b) Nucleotide Sugars 3′ end (a) Polynucleotide, or nucleic acid Deoxyribose (in DNA) Ribose (in RNA) (c) Nucleoside components  3D model of DNA https://www.echalk.co.uk/3Dmolecules/DN A/dna.htm  Hydrophobic bonds within the DNA molecule Hydrophobic bonding is an example of weak van der Waals interactions. There are van de Waals interactions between the stacked planar bases inside DNA A large number of weak van der Waals interactions can significantly increase the stability of a structure, such as the DNA double helix.  “Anti-parallel’ nature of DNA https://www.ncbi.nlm.nih.gov/books/NBK21261/  This is determined by the overall stability of the stacking interactions, which favour right-handed helices. From: https://sandwalk.blogspot.com/2015/03/on-handedness-of-dna.html The major groove carries a “message” that can be read by DNA binding proteins.. Some DNA binding proteins recognise specific nucleotide sequences – so bind at particular region of the DNA 19  DNA has a negative charge.. The backbone of polynucleotides are highly charged (1 unit negative charge for each phosphate group; 2 negative charges per base-pair). important for interaction with histones (positively charged) The negative charge property of DNA can be utilised for laboratory analysis  Importance of counter-ions If there was no salt in the medium surrounding DNA, a strong repulsion between the two strands would exist and the strands would fall apart. Therefore counter-ions are essential for the double- helical structure. Counter-ions shield the charges on the sugar-phosphate backbone.  Alternative forms of DNA A B Z 05/11/2024 24  The predominant form of DNA in vivo is B-DNA But, there is evidence for a role of Z-DNA in vivo: – Z-DNA binding proteins. – Short sections of Z-DNA within a cell are energetically favorable and stable. – Role in regulating gene expression? Base flipping From: Z-DNA in the genome: from structure to disease Ravichandran et al., 2019 DNA can undergo reversible strand separation Denaturation or“melting”of DNA  Effect of temperature on DNA Hydrogen Bonds  Supercoiling of DNA in cells DNA in cells is supercoiled and constrained into loops and this supercoiling and looping influence every aspect of DNA activity. Organisms have evolved DNA supercoiling to toggle between two contradictory requirements: stable and protected DNA, and a readable genetic code. Supercoils form a twisted, 3-D structure which is more favorable energetically. Less stable than relaxed DNA. Negative (left-handed) supercoil: underwound Positive (right-handed) supercoil: overwound All organisms store there DNA as negative supercoiled because this (1) uses less storage space, (2) allows for opening of the helix for transcription, replication etc Supercoiling of DNA 35 RNA can form secondary and tertiary structures  RNA secondary structure Because of the extra hydroxyl group on the sugar, RNA is too bulky to form a stable double helix. RNA exists as a single- stranded molecule. However, regions of double helix can form where there is some base pair complementation (U and A , G and C), resulting in hairpin loops. The RNA molecule with its hairpin loops has a secondary structure. RNA hairpin structure  Conformation of RNA RNA exhibits different conformations: hairpins, stem-loops and tertiary structures such as pseudoknot. Base-paired RNA adopts an A-type double helix 2'-OH group hinders formation of a B-type helix. The folded domains of RNA have catalytic capacities and are called ribozymes. Some of ribozymes can catalyse splicing and self-splicing. RNA helix structure often contains noncanonical base pairs >20 different types of noncanonical (non-Watson-Crick) base pairs. Widen the major groove and make it more accessible to ligands or proteins. 40  Seven major types of RNA Ribosomal RNA (rRNA) Messenger RNA (mRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA) in eukaryotes MicroRNA (miRNA) Piwi-interacting RNA (piRNA) Short interfering RNA (siRNA)  RNA All RNA is synthesized from a DNA template by transcription Multiple kinds of RNA include: Messenger RNA (mRNA) – intermediate form of information from DNA to cytoplasm for processing Ribosomal RNA (rRNA) – class of RNA found in ribosomes, is essential for their function in protein production (translation) Transfer RNA (tRNA) – intermediary adapter molecule between mRNA and amino acids during protein synthesis (translation) © McGraw Hill, LLC 42 Fig. 17-3 TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell  Ribosomal RNA (rRNA) Functions as structural and enzymatic component of ribosomes 80% of total cellular RNA Region in nucleus where rRNA is transcribed is the nucleolus It has sequence complementarity to regions of the mRNA so that the ribosome knows where to bind to an mRNA it needs to make protein from 45 Messenger RNA (mRNA) Acts as intermediate between genetic code of DNA and amino acid sequence of proteins 3% of total cellular RNA 47 Ribosome binding site on mRNA © McGraw Hill LLC. All rights reserved. No reproduction or distribution without the prior written consent of McGraw Hill LLC. 49  Transfer RNA (tRNA) Adapter molecule that carries amino acids to specific codon on mRNA in ribosome during protein synthesis 73-95 nucleotides long 5% total cellular RNA 50 Modified nucleotides in tRNA  tRNA Binding site for 3’OH amino acid H bonds to complementary base pairs 5’ 1. 5′ and 3′ ends 2. hydrogen bonds 3. unpaired regions 4. anticodon loop 5. binding site for amino acid Unpaired regions Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Anticodon loop  Figure 15.14 © McGraw Hill LLC. All rights reserved. No reproduction or distribution without the prior written consent of McGraw Hill LLC. 53  Multiple kinds of RNA All RNA is synthesized from a DNA template by transcription Multiple kinds of R N A include: Small nuclear RNA (snRNA) – part of machinery involved in processing of “pre-mRNA” in splicing Signal recognition particle RNA (SRPRNA) – mediator for proteins synthesized on the rough endoplasmic reticulum Small RNAs (miRNA and siRNA) – involved in control of gene expression 55  Summary Natural cellular DNA (B DNA) contains two complementary anti-parallel polynucleotide strands wound together into a regular right-handed double helix with the bases on the inside and the two sugar- phosphate backboned on the outside. This DNA supercoiled (predominantly negative) to allow for storage and gene activity  Summary There are at least 7 major types of RNA. The shape of RNA structure depends on the bases present, and includes non-standard bases and non-Watson-Crick bonding. Some RNA molecules are involved in the process of gene expression, others have enzymatic functions – related to the structure of the molecule 05/11/2024 57 MCQ quiz for Lecture 12: Nucleic Acid Structures Answers will be given in your Seminar sessions – with further discussion. You must attempt before your seminar session. These quizzes are part of your learning for the Biochemistry module They will aid your on-going studies at the University of Westminster  1) Chargaff’s Rules state that… a) DNA is the information carrying molecule. b) Purines hydrogen bond to pyrimidines. c) The amount of adenine in a genome is equal to the amount of uracil. d) The amount of cytosine in a genome is equal to the amount of guanine. e) The ratio of purine to pyrimidines is the same across species. 2) The structure of genomic DNA in vivo is _____. a) Mainly a right-handed double helix in the B form. b) Mainly a left-handed double helix in the B form. c) Never found in the Z form. d) Mainly a right-handed double helix in the A form. e) Always a right-handed double helix in the B form. 3) The structure of mammalian RNA in vivo is ____. a) Always a single long, linear strand. b) Always a stable double helix forming complex shapes. c) Usually exists as a ‘T’ shape as in transfer RNA. d) Can contain base pairs different from the standard Watson- Crick pairing. e) Random coils with no fixed structure. 4) The stability of DNA in vivo partially depends on____. a) Interaction with specific enzymes in the nucleus. b) Having a positively charged phosphate backbone. c) Hydrophobic interactions with surrounding molecules. d) Being able to flip into the z form structure. e) The presence of cationic counter ions. 5) Which of the following statements is incorrect? a) DNA cannot renature after heating. b) The Tm of a strand of DNA refers to the temperature when it is 50% denatured to single strands. c) The structure of DNA was determined by the technique of X ray crystallography. d) Some forms of RNA can act as biological catalysts. e) In a genome A =T and C = G.  snRNA Small nuclear RNA, in eukaryotes Involved in removal or splicing of introns from eukaryotic mRNA Catalytic or associated with catalytic proteins 05/11/2024 64  miRNA Micro RNA Small RNA (~22 nucleotides), often complementary to untranslated region of mRNA Involved in regulating gene expression ~1,000 in human genome, regulating ~60% of genes 05/11/2024 65  piRNA Piwi-interacting RNA 26-31 nucleotides long Largest class of mammalian small RNAs ‘Guardians of the germline’ Predominantly found in the male germline Bind to Piwi proteins Role in control of transposable elements (short transferable sections of DNA) Also role in gene silencing 05/11/2024 66  siRNA Short interfering double-stranded RNA molecules Usually 21 bases long with an overhang of two at each 3’ terminus Recruits RNA-induced silencing complex (RISC) for mRNA cleavage and gene silencing Used experimentally to knock down specific gene expression 05/11/2024 67  Ribozymes 05/11/2024 68