Lec 6.1-Nucleic Acids.pptx

Biochemistry 1/Chemistry 121

Biochemistry 1/Chemistry 121Biochemical Molecules-Nucleic AcidsQuestions?QuestionsHow do lipids differ from other biological molecules?What types of lipids are important for membranes in brain tissue?Double bonds in unsaturated fatty acids are usually what?How is chain length and saturation of fatty acids related to their melting point? QuestionsVitamin D is derived from what?What do phospholipases do?What are the five categories of steroid hormones?Which group of lipids is derived from a five-carbon subunit?

Nucleic AcidsNucleic AcidsDNA (Deoxyribonucleic Acid)RNA (Ribonucleic Acid)Free nucleotides-The subjects today are:+DNA (Deoxyribonucleic Acid)+RNA (Ribonucleic Acid)+Free nucleotidesEach plays a central role in biochemistry

6Central Paradigm or DogmaDNA makes RNA makes ProteinDNAGenes and the GenomeNuclear vs. OrganellarRNATranscriptionCoding & Non-codingProtein TranslationMulti-functional-DNA makes RNA makes Protein+This is the central paradigm of molecular biology as elucidated by Marshall Nirenberg and colleagues is "DNA makes RNA makes Protein. There are occasional and important exceptions, but this is the central dogma of biology.-DNA+Genes and the Genome+Nuclear vs. Organellar+The DNA of eukaryotic cells is in two parts-nuclear and organellar. The vast majority of genes are contained in the nuclear genome, but important components of the genome are carried by organelles, namely the mitochondria (and the chloroplasts of plants, but we wont be going there). Organellar inheritance (through the mother) is an important consideration, but not for this course in biochemistry.-RNA+Transcription+Coding vs. Non-coding+The RNA of cell is important for both transcription and translation, but we usually only think of it in terms of transcription. With RNA, there are two very different pools of molecules-the coding or mRNA and the much larger group of non-coding RNA. More on this later-Protein+Translation+Multi-functional+Proteins are the product of the transcription of DNA to RNA then the translation of RNA to protein. While RNAs have many functions, it is the proteins that make up the bulk of the cells machinery and infrastructure along with lipids and other molecules.

7Basic Structure of Nucleic AcidsNitrogenous BasePurines PyrimidinesPentose Sugar2-deoxyriboseRibosePhosphate Group-There are three components of the nucleic acids-Nitrogenous Bases+Purines +Pyrimidines-Pentose sugar+2-deoxyribose+Ribose-Phosphate group+This can be one, two, or three phosphate groups+In polynucleotides, there is only a single phosphate group between each +We will visit a concept of high-energy molecules today as well as the phosphate groups of molecules such as adenosine triphosphate are the energy currency of life.

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Nitrogenous Bases -These are the skeletons of the purines and pyrimidines-The purines, Adenine and Guanine. Obviously these are double-ringed structures again attached to the 1 Carbon atom, but through the 9-N position.-The pyrimidines, Cytosine, Thymine, and Uracil. These are six membered rings attached to the 1' Carbon atom of the sugar.-I would know these structures. It is important to note the substructure differences between the two molecules. They differ in their H-bonding potential

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Nucleotide Nomenclature-I would know these structures. It is important to note the substructure differences between the molecules. They differ in their H-bonding potential-We will go into the H-bonding potential later today.

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Carbohydrate moieties-The two sugars used in nucleic acids are ribose and its close cousin 2-deoxyribose.-This is a Haworth projection of the 5-Carbon sugar.-Ribose is the structural sugar for RNA whereas 2-deoxyribose is the structural sugar for DNA, hence the name.

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Nucleotide structure-When you put together the three parts, you get the complete nucleotide.-The more prevalent form of the nucleotide is the 5-nucleotide shown in panel A-Nucleotides with a phosphate just at the 3 position arent normally found in nature, but the 3 linkage is an important part of the structure when we get into polynucleotides.

12DNAChief function is as the master plan of us2-deoxyribose sugarNitrogenous BasesAdenine (purine)Thymine (pyrimidine)Guanine (purine)Cytosine (pyrimidine)Phosphate Group-As mentioned earlier, DNAs chief function is as the master plan of us-It uses the 2-deoxyribose sugar-It uses the four nitrogenous bases+Adenine+Thymine+Guanine+Cytosine-Lastly, a Phosphate group

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Nucleic Acid Polymer-Moving from individual Nucleotides to DNA polymers. Individual nucleotides are linked together by phosphodiester bonds. The linkages between the individual nucleotides is 5 to 3. This results in chemically distinct ends.-The 5 end has an unreacted triphosphate group while the 3 end has a free hydroxyl group. In addition, the backbone of the DNA polymer has a distributed negative charge making it ionic.-This is the basic polymer structure of a nucleic acid-The base structure shown here is that of RNA with the sequence AUCG.-The Polymer is read 5 to 3, just as it is synthesized-The synthesis is energetically driven by the hydrolysis of the triphosphate form of the single nucleotide to extend the chain by one more nucleic acid and leave a molecule known as pyrophosphate-Pyrophosphate is subsequently hydrolyzed to two phosphate molecules-In RNA, Uracil replaces Thymine-Note the other key structural difference of the 2 hydroxyl group seen in RNA, but not in DNA-This structural difference does play a role in the function of RNA, but it is not something we will delve into in this course

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DNA Nitrogenous Bases-For the most part, DNA does not exist in single stranded form (there are exceptions to this, but we wont go into them).-Base pairing is the first major characteristic of the double helix to consider. This involves base pairing between Adenine and Thymine and between Cytosine and Guanine. As I pointed out earlier, there are structural characteristics of the nitrogenous bases that point to the H-bonding patterns.-Guanine has a carbonyl, a free amino group, and a nitrogen.-Cytosine is almost a mirror image with a carbonyl, a free amino group, and a nitrogen.-In the case of Adenine, there is a free amino group -Thymine has a nitrogen with an exchangeable hydrogen and a carbonyl group that is accessible for H-bonding

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DNA base pairingBase pairing is the first major characteristic of the double helix to consider. This involves base pairing between Adenine and Thymine and between Cytosine and Guanine. As I pointed out earlier, there are structural characteristics of the nitrogenous bases that point to the H-bonding patterns

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DNA Nitrogenous Bases-The next major characteristic is base stacking, also call - interactions or hydrophobic interactions. If we go back and look at the base structure, we see that all three have extensive -bond, or double bond structure. The double bonds are actually delocalized and shared by all the atoms in the ring.-When -bonds form into a sandwich, they like to stick together and the structure becomes more stable, the better the stacking. This - interaction helps to further stabilize the DNA double helix.

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DNA Double Helix-This figure helps to summarize much of what was just described. The figure on the right shows the H-bonding of the base pairs. On the left, you see a view of the stacking that goes on in a double helix. I havent mentioned the geometry the the double helix, but it is important too.-First the helix is right-handed, next we have the Major groove and the minor groove. The major groove is wide and deep while the minor groove is narrow and relatively shallow. Allows the machinery of replication and transcription to access the genetic code.-It is important to note that the base-paired strands run in opposite directions, but both are 5 to 3

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DNA Major/Minor Groove-DNA binding proteins need to interact with specific sequences of DNA-They can do this via interactions such as H-bonding and van der Waals interactions (hydrophobic)

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-This slide is a color-coded view of how DNA binding proteins can selectively recognize specific stretches of DNA through H-bonding and vdW interactions-Contacts between the DNA & protein are non-covalent with H-bonds being the dominant force in the major groove and hydrophobic interactions in the minor groove. On the surface phosphodiester backbone interacts electrostatically. Only generalization to be made as the details of each proteins interaction with the DNA is unique.

20Key Features of DNARight-handedStrands run in opposite directionsExtensive hydrogen bonding across strandsBase pairingA::T (two H-bonds)C:::G (three H-bonds)Base stacking (aka p-p interactions or hydrophobic interactions)Structurally flexible-DNA is right-handed-Strands run in opposite directions-Extensive hydrogen bonding across strands-Base pairing+A:T (two H-bonds)+C:G (three H-bonds)-Base stacking helps to stabilize the structure-Structurally flexible

21RNAMulti-functionalCoding (Transcription)Non-coding (Multi-faceted)Ribose sugarNitrogenous basesAdenine (purine)Uracil (pyrimidine)Guanine (purine)Cytosine (pyrimidine)Phosphate group-Multi-functional+Coding (Transcription)+Non-coding (Multi-faceted)-Ribose sugar instead of 2-deoxyribose-Nitrogenous bases+Adenine (purine)+Uracil (pyrimidine instead of thymine)+Guanine (purine)+Cytosine (pyrimidine)-Phosphate group

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RNA vs. DNA-While this is yet another picture of a ribose vs. a deoxyribose, focus on the phosphate backbone-These are triphosphate nucleotides.-We will talk more about these later today, but these are considered high-energy forms of the nucleotide

23RNACoding RNA (95%)rRNA (ribosomal)tRNA (transfer)snRNA (small nuclear)snoRNA (small nucleolarmiRNA & siRNA (micro & short interfering)-We mentioned earlier that DNA is the stuff that codes for inheritance, but what does RNA do.-RNA has many functions. +The first function that comes to mind is in translation, communicating what the genome wants into a protein. mRNA is synthesized in the nucleus then transported out of the nucleus (in eukaryotes) and used as a template for protein synthesis. In Prokaryotes, the mRNA is not transported out of the nucleus, as there is no nucleus. The mRNA pool is actually further subdivided in eukaryotes with pools of coding RNA, Pre-RNA, and mature RNA.+rRNA helps form part of the machinery of protein synthesis (translation). rRNA is a key structural and catalytic component in protein synthesis+tRNA is also an important component of the protein synthesis machinery, but its role is in translating the mRNA into protein. tRNAs also contain derivatives such as hypoxanthine (inosine) and pseudouridine. These modifications are made post hoc after the original tRNA is synthesized from the template.+snRNA (small nuclear RNA) is Uracil-rich and closely tied to the processing of mRNA. As I mentioned, the mRNA pool is actually composed of smaller pools of mRNA that must be processed by the cell into mature mRNA and the snRNA are involved in that process+snoRNA (small nucleolar RNA) is involved with the processing of rRNA+miRNA and siRNAs are involved with the regulation of gene expression and are still not that well understood

24tRNA Structure

-tRNA has four components+The "Acceptor arm" with the invariant sequence 5'-CCA-3'+The "D arm" with dihydrouridine+The "anticodon arm" with the nucleotide triplet which will base pair with the mRNA during translation+The "V loop" which is 3-5 nucleotides in Class 1 tRNAs and 13-21 in Class 2 tRNAs+The "TPsiC arm" with an invariant thymidine-pseudouridine-cytidine-The physical structure is drawn in the cloverleaf form for 2D consumption. -Vertebrate mitochondrial tRNAs sometimes lack parts of this structure, such as tRNAserine in human mitochondria which is missing the "D arm."-The accepted nomenclature for tRNAs is +tRNAGly1+tRNAGly2-The A, C, G, T, U, Psi labeled in this structure are invariant, meaning they are found at the same positions in every tRNA.-The labeled R & Y (Purines and Pyrimidines) are semi-variant-There are 5-10 nucleotides that are chemically modified in any given tRNA

25tRNA structure

-The actual 3D structure is more akin to an "L" with the "Acceptor arm" at one end and the "Anticodon arm" at the other end.-It will be important to note in a few minutes that the three nucleotide "anticodon" is not completely flat, or straight.-The elaborate base stacking and pairing adds to the overall stability of the structure

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Central Dogma-Thus far we have spoken about the structure of DNA and RNA, but how do these result in the production of proteins.-We are only going to touch upon this subject as it is worth noting, but an in depth look at the subject is a course by itself.-We also wont go into the special cases where the RNA makes RNA and RNA is used to make DNA

27Genes and Gene ExpressionGenesGene StructureProkaryotes vs. Eukaryotes-We will first talk about genes then move onto briefly talk about Gene Structure and lastly we will touch upon the differences between prokaryotes and eukaryotes

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-Some of you might be wondering why we will touch upon prokaryotes.-There is a growing realization that the microbiome, as it is called is extremely important in understanding the proper functioning of the human body.-We dont have time to really dive into the subject in this course, but I hope you will have a chance in your other courses to develop a better understanding of the almost symbiotic relationship we have with some microbes.-There have been advances in treating disease when the microbiome is taken into account and I firmly believe there will be even more advances in the future that will help improve patient quality of life

29What is a gene?A DNA segment containing biological information and hence coding for an RNA and/or polypeptide moleculeAnatomy of a geneOpen Reading FrameIntrons vs ExonsUpstream Regulatory Sequences-A DNA segment containing biological information and hence coding for an RNA and/or polypeptide molecule-Anatomy of a gene+Open Reading Frame (this part of the gene is what will eventually result in a protein or RNA being synthesized++Introns vs Exons (This is a key facet of eukaryotic genes, but is absent in prokaryotic genes)+Upstream Regulatory Sequences (We will talk a little about upstream regulatory sequences. There are also downstream regulatory sequences that we wont touch upon as they are rather convoluted and could take up an entire lecture)

30Anatomy of a Gene

-This is a translation table for translating DNA (or RNA) sequence into protein-Every three nucleotide corresponds to a codon, each codon selects for a particular amino acid or a stop signal-The important thing to also note is that there is only one codon that codes for the amino acid methionine. This codon also serves as the start signal for beginning protein synthesis (translation). This does not mean that there arent methionines present at other positions in a protein, but provides us with a starting point.

31Prokaryotic Gene

-This shows the basic structure of a prokaryotic gene.-There is a sequence upstream of the gene termed the promoter-The promoter is where machinery of transcription will bind to the DNA and begin making mRNA-The mRNA begins before the start codon as there are regulatory sequences carried within the mRNA that the translation machinery needs -Prokaryotic genes are organized into operons where several genes will be strung together in a single message, but coding for different proteins. These proteins are usually all part of the same biosynthetic pathway, but not all members of the pathway will be in the same operon.-The organization of operons is often related to how the pathway is controlled

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Eukaryotic Genes-As I mentioned, eukaryotic genes are made up of introns and exons-Exons code for the actual protein whereas introns do not-The introns are processed out of the mRNA while they are still in the nucleus of the cell-This cassette-like structure has all sorts of implications that can result in differential expression of proteins-Namely, a single gene can code for more than one protein as there can be exon shuffling or alternative splicing-There are diseases where there is defective gene splicing, such as cystic fibrosis and the Cystic fibrosis conductance transmembrane regulator (CFTR) gene

33Eukaryotic Gene

-As with prokaryotic genes, there are regulatory sequences controlling the initiation of transcription.-You dont need to know what these sequences are, just know that there are promoters that control gene expression in all organisms

34SummaryDNA and RNA are linear polymers composed of a 5-Carbon sugar, a nitrogenous base, and a phosphate groupDNA uses adenine, guanine, thymine, and cytosineRNA uses adenine, guanine, uracil, and cytosineBoth DNA and RNA run 5 to 3The DNA double helix has a major and a minor groove that also provides sequence identity-DNA and RNA are linear polymers composed of a 5-Carbon sugar, a nitrogenous base, and a phosphate group-DNA uses adenine, guanine, thymine, and cytosine-RNA uses adenine, guanine, uracil, and cytosine-Both DNA and RNA run 5 to 3-The DNA double helix has a major and a minor groove that also provides sequence identity

35SummaryGenes in eukaryotes have introns and exons whereas prokaryotes are organized into operons without intronsIn eukaryotes, the genome is split between the nucleus and the organellesHumans have roughly 20,000 genesThe human genome has roughly 3 billion base pairsThere are significant amounts of non-coding DNA (about 98%) in humans

This summarizes the central Dogma of Molecular Biology 37High Energy Compounds-Why did we spend all this time talking about nucleotides?-Besides their role in inheritance and transcription/translation, they are used extensively in the cell as an energy currency

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ATP & ADP-The most frequently encountered high-energy compound is ATP, adenosine triphosphate-For ATPs proper function, Mg2+ is an absolute requirement-Why is Mg2+ required? The reason is in the triphosphate portion of the molecule. The three negative charges put considerable strain on the molecule and without the Mg2+ the negative charges would cause the ATP to bind to other molecules with a net positive charge.-Why is ATP considered high energy, but ADP not? This has to do with resonance stabilization. ADP and Pi are more resonance stabilized than ATP so the conversion of ATP to ADP lowers the energy of the system (diagram resonance). Resonance stabilization of phosphoanhydride bond is less than that of its hydrolysis product. Electronic repulsion between charged groups of the phosphoanhydride. At physiological pH, ATP has 3 to 4 net charges. Lastly, differences in solvation energy-AMP is an important regulatory molecule that we will see later when we get into metabolism-In addition to ATP, GTP is also an important energy molecule and an important regulatory molecule that we should encounter when we get to the topic of signal transduction in BC2.

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Activation of Carbohydrates-In addition to nucleotides role in energy currency through ATP and GTP, ATP and other nucleotides are important activators for polysaccharide biosynthesis

40Questions?QuestionsWhat is the Central Dogma of Molecular Biology?What are the components of a nucleotide?What is the structural difference between DNA and RNA?What are Purines?What are Pyrimidines?QuestionsWhat forces stabilize the double helix of DNA?How many H-bonds does the G:C pair make?How many H-bonds does the A:T pair make?What percentage of RNA is coding in humans?What percentage of DNA is coding in humans?QuestionsWhat are the functions of AMP, ADP, and ATP?Why is ATP considered a high-energy compound?What element is important for the proper function of ATP?