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BIOCHEMISTRY COURSE – Pharmacy Biomedical Preview program, Summer 2017 Patrick Fotso, B.S. Biochemistry University of Maryland Baltimore County (UMBC) 2nd Year Pharmacy Student Contact: firstname.lastname@example.org Phone: 2404292374
WHAT IS BIOCHEMISTRY? Biochemistry = Biology + Chemistry Biology is the “What” Chemistry is the “Why” Biochemistry is the “How” It explores chemical processes within living organisms
1. OVERVIEW OF NUCLEIC ACID CHEMISTRY DNA Structure & Components RNA Structure & Components Other Functions of Nucleotides
WHY ARE NUCLEOTIDES & NUCLEIC ACIDS IMPORTANT? The structure of every biomolecule and cellular component is a product of information programmed into the nucleotide sequence of a cell’s nucleic acids.
NUCLEOTIDES • Nucleotides have three characteristic components: • a nitrogenous (nitrogen-containing) base, • a pentose, • a phosphate (G) (A) (U) Bases of DNA (C) (T)
NITROGENOUS BASES: MNEUMONIC Your heart is PURe as AG Purines are A & G; Ag = chemical gold; double-ring = heart
NUCLEOTIDES& NUCLEIC ACIDS • Discovery of the structure of DNA by Watson and Crick in 1953 • Nucleotides are the constituents of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) • A segment of a DNA molecule that contains the information required for the synthesis of a functional biological product, whether protein or RNA, is referred to as a gene. DNA is a Double Helix
NUCLEOTIDES The base of a nucleotide is joined covalently (at N-1 of pyrimidines and N-9 of purines Nucleic acids have two kinds of pentoses; 1. The recurring deoxyribonucleotide units of DNA contain 2’-deoxy-D-ribose 2. The ribonucleotide units of RNA contain D-ribose.
DNA & RNA The successive nucleotides of both DNA and RNA are covalently linked through phosphate-group “bridges,” in which the 5-phosphate group of one nucleotide unit is joined to the 3-hydroxyl group of the next nucleotide, creating a phosphodiester linkage. Oligonucleotide – a short nucleic acid Polynucleotide – a longer nucleic acid
DNA • Deoxyribonucleic acids (DNA) • Adenosine, Guanine, Cytosine, Thymine • A&T pair, C&G pair • A-T has 2 hydrogen bonds • C-G has 3 hydrogen bonds • Super-coiled strands of DNA polymers are stored as chromosomes in the nucleus of every cell • Genes are sections of DNA that determine hereditary traits by transcription • Portions that are transcribed (used) are “Coding Portions” • DNA that is not transcribed (used) are “Non-Coding Portions” • Recent studies determined that only 8.2% of our entire genome is transcribed and used; the vast majority is junk coding
DNA Hydrophobic stacking interaction of bases minimizes contact of water with bases. Its important for stabilizing the DNA structure A bonds specifically to T (or U) and G bonds to C are two types of base pairs that predominate in double-stranded DNA and RNA,
DNA Shape Complementarity A. Compatible B. Incompatible
DNA PAIRING DNA is formed of two bonded strands • Double Helix form • 1 strand can code for the other “complementary” strand DNA is read 5’ to 3’ The complementary strand is sequenced via DNA pairing Ex: 5’ AAATTGCGCGC 3’ TTTAACGCGCG Swap it so it also reads 3’-5’: GCGCGCAATTT
CHECK POINT QUESTION What is the complementary sequence of 5’ AAGTCCCAAGT 3’ A.) 3’ GGCAGGGTTAA 5’ B.) 3’ TTCTGGGAAGT 5’ C.) 3’ TTCAGGGTTCA 5’ D.) None of the Above
DNA STORAGE • DNA is coiled around histones • structural/organizational proteins • DNA-wrapped histones supercoil further into chromosomes • 23 pairs of chromosomes are located in the nucleus of every cell • Every cell contains around 1 meter (3 feet) of genome, supercoiled to fit into a nucleus!
RNA • Ribonucleic acids (RNA) • Adenosine, Guanine, Cytosine, Uracil • A&U pair, C&G pair • A-U has 2 hydrogen bonds • C-G has 3 hydrogen bonds • Single-stranded (except in the case of tRNA) • RNA serves several purposes • mRNA: Messenger RNA • The “recipe” for proteins. Fed through and read by ribosomes, where amino acids are assembled into proteins according to the letter-codes • rRNA: Ribosomal RNA • Makes up ribosomes • tRNA: Transfer RNA • Finds amino acids and brings them to the ribosomes
PREDICTING RNA FROM DNA 5’ AGTTGCGCATATAT 3’ Complementary DNA: 3’ TCAACGCGTATATA 5’ Complementary RNA: 3’ UCAACGCGUAUAUA 5’ (Oriented 5’-3’: AUAUAUGCGCAACU)
RNA RNA chains frequently fold back on themselves to form base-paired segments between short stretches of complementary sequences. Factors affecting stability of RNA double helical structure: The additional, non-Watson-Crick base pair. This is the G:U base pair. Base-stacking interactions in the loop RNA Can Fold Up into Complex Tertiary Structures
Other Functions of Nucleotides • Nucleotides also function as; • Energy carriers • Components of enzyme cofactors • Chemical messengers Energy carriers Hydrolysis of nucleoside triphosphates provides the chemical energy to drive a wide variety of cellular reactions. Adenosine 5-triphosphate, ATP, is by far the most widely used for this purpose, but UTP, GTP, and CTP are also used in some reactions.
CHECK POINT QUESTION Which of the following is not a function of Nucleotides? A.) Energy carriers B.) Components of Nucleic Acids C.) Chemical Messengers D.) All of the Above E.) A & B only
Other Functions of Nucleotides Components of enzyme cofactors A variety of enzyme cofactors serving a wide range of chemical functions include adenosine as part of their structure eg.
Other Functions of Nucleotides Chemical messengers or Regulatory Molecules Cells respond to their environment by taking cues from hormones or other external chemical signals. The interaction of these extracellular chemical signals (“first messengers”) with receptors on the cell surface often leads to the production of second messengers inside the cell. Often, the second messenger is a nucleotide. One of the most common is adenosine 3,5-cyclic monophosphate ( cyclic AMP or cAMP), which is synthesized from ATP . cAMP
2. CENTRAL DOGMA: DNA, RNA, & PROTEIN BIOSYNTHESIS DNA Synthesis & Replication Transcription Translation
CENTRAL DOGMA OF MOLECULAR GENETICS • DNA is the central repository for genetic information in the cell. But DNA itself does not directly regulate the synthesis of proteins. • The information carried by DNA is copied into an intermediate form, messenger RNA (mRNA). • mRNA is used as a template to direct the proper assembly of amino acids into the protein gene product. • DNA does act as a template for its own replication. 5’ 3’ 5’ 3’ 5’ 3’ COOH H2N
CHECK POINT QUESTION What type of RNA is used as a template to direct protein synthesis? A.) rRNA B.) mRNA C.) tRNA D.) snRNA
THE CENTRAL DOGMA: DNA, RNA, PROTEINS • The Recipe Metaphor: • DNA: Cook Book • RNA: Recipe • Proteins: Cake • Proteins make up the majority of our body • Structure, enzymes • DNA ➔ RNA ➔ Proteins
DNA REPLICATION Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand. This is semiconservative replication. A new strand of DNA is always synthesized in the 5’ to 3’ direction, with the free 3’ OH as the point at which the DNA is elongated.
DNA REPLICATION DNA is synthesized by DNA Polymerase
DNA REPLICATION Two central requirement for DNA Polymerization: • DNA polymerase requires a template. • Polymerase requires a Primer. A primer is a strand segment (complementary to the template) with a free 3-hydroxyl group to which a nucleotide can be added.
CHECK POINT QUESTION In what direction is DNA newly synthesized? A.) 5’ 3’ B.) 3’ 5’ C.) Both directions
DNA REPLICATION The synthesis of a DNA molecule can be divided into three stages: Initiation = Opening of the DNA helix at the origin to establish a pre-priming complex for subsequent reactions via helicases. Elongation = Leading strand synthesis and lagging strand synthesis via DNA polymerase. Termination = Ligase joining the growing strand of Okazaki fragments.
DNA REPLICATION DNA is degraded by nucleases, or DNases Different nucleases, belonging to two broad classes: • Exonucleases - degrade nucleic acids from one end of the molecule, removing nucleotides only from the 5’ or the 3’ end. • Endonucleases - can begin to degrade at specific internal sites in a nucleic acid strand or molecule, reducing it to smaller and smaller fragments.
DNA REPAIR A permanent change in the nucleotide sequence of DNA is called a mutation. Mutations can involve the replacement of one base pair with another (substitution mutation) or the addition or deletion of one or more base pairs (insertion or deletion mutations). If the mutation affects nonessential DNA or if it has a negligible effect on the function of a gene, it is known as a silent mutation. Repair Systems • Mismatch Repair • Base-excision Repair • Nucleotide-excision Repair • Direct Repair
Transcription is a process by which DNA acts as a template for the synthesis of a complimentary RNA strand TRANSCRIPTION OF RNA • Like replication, transcription has initiation, elongation, and termination phases, however initiation is further divided into discrete phases of DNA binding and initiation of RNA synthesis • Transcription differs from replication in that it does not require a primer and, generally, involves only limited segments of a DNA molecule. Additionally, within transcribed segments only one DNA strand serves as a template.
TRANSCRIPTION OF RNA RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3’-hydroxyl end, building RNA in the 5’ to 3’ direction.
STAGES OF TRANSCRIPTION Initiation & Elongation • In the binding phase, first the RNA polymerase binds to the promoter, forming, in succession, a closed complex (in which the bound DNA is intact) and an open complex (in which the bound DNA is intact and partially unwound. • Second (Initiation phase), transcription is initiated within the complex, leading to a conformational change that converts the complex to the elongation form, followed by movement of the transcription complex away from the promoter (promoter clearance). • Polymerase leaves the promoter and becomes committed to elongation of the RNA in the elongation phase. Termination Termination – release of RNA polymerase from DNA and termination of chain extension
DRUGS THAT INHIBIT TRANSCRIPTION • DNA intercalating agents: • Flat molecules that squeeze between adjacent base pairs • Doxorubicin • DNA alkylating agents: • Add bulky hydrocarbon chains or groups to DNA • Cyclophosphamide
HOW ARE PROTEINS SYNTHESIZED The tRNA “translates” the nucleotide sequence of an mRNA into the amino acid sequence of a polypeptide. Translation – is the process of mRNA-guided protein synthesis The Genetic Code Codons are the key to the translation of genetic information, directing the synthesis of specific proteins. Codon is a triplet of nucleotides that codes for a specific amino acid CCU: Proline AGU: Serine Important codons • Initiation codon AUG • Termination codons (UAA, UAG, and UGA)
CHECK POINT QUESTION Which of the following is not a STOP codon? A.) UGA B.) UAA C.) UAG D.) UGG
TRANSLATION • Process by which proteins are synthesized from the sequence encoded by mRNA • Initiation – recruitment of ribosome to a mRNA • Elongation – synthesis of amino acid chain using codon template • Termination – release of completed protein from ribosome
CHECK POINT QUESTION Codon templates are used in which part of Translation? A.) Initiation B.) Elongation C.) Termination D.) None of the Above
RNA IS TRANSLATED TO PROTEINS • Proteins are polymers of amino acids • mRNA is fed through ribosomes and amino acids are joined together according to the sequence of RNA