Biochemistry III Information PDF

Biochemistry III information Textbooks Lehninger. Principles of Biochemistry, 8th ed. Cox. Molecular biology, Principles and practice, 2nd ed. ILIAS: 2003-HS2024-0: Biochemie III Exam: 13. Jan 2025, 13:00 - 15:00: EG16 Contact: [email protected] [email protected] oliver.muehlemann@uni...

Biochemistry III information Textbooks Lehninger. Principles of Biochemistry, 8th ed. Cox. Molecular biology, Principles and practice, 2nd ed. ILIAS: 2003-HS2024-0: Biochemie III Exam: 13. Jan 2025, 13:00 - 15:00: EG16 Contact: [email protected] [email protected] [email protected] 1  How to study Biochemistry (III) Terminology / Jargon language / Accurate description Do I use the right Biological role/significance terms to describe Why does it Where/When? the mechanism? happen? Is it Is the process necessary for cell relevant to all survival? How organisms/cells? much energy Does it take place does it require and in a specific when? compartment? Look for unifying principles: similarities, differences and repetitive concepts  How to study Biochemistry (III) What is the biological importance? There may be different roles for the same process, in some cases, one is more important and was probably the first during evolution. Is it the process necessary for cell survival? If so, this is probably associated with great expenses in energy. Take some time to understand which process steps consume energy and how this energy is consumed. Necessary for enzymatic activities? Proofreading? Conformation change? What is the importance of the experiments and methods that are described?  How to study Biochemistry (III) What “common features” are used in different mechanisms? For example, molecular mimicry, inhibition caused by conformational changes etc. What is required for a process to be performed? This knowledge allows the in vitro reconstitution of a process and understanding an experimental design. Enzymes, specific proteins, cofactors, do not forget energy source if required. What is the role of the same enzymatic activity in different processes? For example, processes that require nucleolytic activities.  5 Biochemistry 1. The role of proteins in DNA and III RNA metabolism - DNA Structure Evangelos Karousis, PhD Website Dept. of Chemistry, Biochemistry and Pharmaceutical Sciences University of Bern  Learning goals The role of proteins in DNA and RNA metabolism DNA Structure 01 02 03 Introduction: Define and Explain and predict the Describe the structural distinguish steps of gene role of proteins in RNA features of nucleic acids expression and DNA metabolism and explain their function: special focus on hybridization and their role as enzyme templates  Information flow in biological systems Alberts, 7th edition Fig.1.5 Alberts, 7th edition Fig.1.4  Information flow in biological systems Transcription: DNA to RNA, RNA polymerase on DNA template in the nucleus Translation: mRNA to protein, ribosomes on mRNA template in the cytoplasm. In procaryotic cells, all processes occur in the cytoplasm How do small things compare to each other? Biorender What is bigger? a. A protein b. The mRNA that encodes it 10 What is bigger? a. A protein b. The mRNA that encodes it 11 Cells in textbooks … vs … Cells in (almost) real life 10.2210/rcsb_pdb/goodsell-gallery-001 David S. Goodsell Proteins and nucleic acids Packaging: structural role (proteins) Catalysis: enzymes (proteins and some RNAs) Movement: mostly proteins, some RNAs recruit other molecules 13  Protein folding principles The presence or absence of a protein confers a property Structure is essential for function. The amino acid sequence (primary structure) defines the protein shape Hydrophobic interiors. Polar amino acids: outer surface. Protein domains are modular units found in different proteins Alberts, 7th ed. Fig. 3-5 Some proteins bind other molecules Ligands: molecules bound by a protein (nucleic acids or other proteins). Reversible. One exception in DNA repair. Binding site is complementary in size, shape, charge, hydrophobic state Conformational flexibility Induced fit: permits tighter binding of a ligand Cooperativity: one subunit enhances binding of other subunits Regulation: Adjustment by other ligands and protein modifications Enzymes in gene expression DNA and RNA metabolism Genetic diseases - drug targets Biotechnology tools Substrate: the target molecule of an enzymatic reaction, it is changed (difference to ligands) Genomes 4, T. A. Brown Fig. 2.4 Enzymes in gene expression Activities Enzyme Function DNA Polymerase Synthesizes new DNA strands using a DNA template. Polymerases RNA Polymerase Synthesizes RNA from a DNA template during transcription. Reverse Transcriptase Converts RNA into complementary DNA (cDNA) - Telomerases Restriction Enzymes Cuts DNA at specific recognition sites, producing fragments. Removes nucleotides from the ends of DNA molecules, important in DNA Exonuclease repair. Endonuclease Cleaves bonds within a DNA strand, involved in repair and recombination. Nucleases Degrades RNA by cleaving phosphodiester bonds, important in RNA Ribonuclease (RNase) processing. A CRISPR-associated endonuclease that cuts DNA at specific sites for genome Cas9 editing. Joins DNA fragments by forming phosphodiester bonds, essential in repair Ligases DNA Ligase and replication. Unwinds the double-stranded nucleic acids, removes proteins from nucleic Helicases Helicase acids Topoisomerases Topoisomerase Affect supercoiling in DNA by cutting and rejoining DNA strands. Hydrolases ATPase/GTPase Hydrolyse GTP to GDP, important regulatory consequences DNA Methyltransferase Adds methyl groups to DNA, affecting gene expression. Modification Kinase Adds phosphate groups to molecules, such as nucleotides or proteins. Phosphatase Removes phosphate groups from DNA, RNA, or proteins.  DNA and RNA binding proteins Protect, organize, regulate, and mediate nucleic acid metabolism Nonspecific: irrespective of sequence - electrostatic interactions with negatively charged phosphate groups. SSB (Single-stranded DNA-binding protein) Specific: tight binding on target sequences - hydrogen bonds with specific base pairs. Most regulators of gene expression 18  DNA and RNA binding proteins Binding size (n): The number of nucleotides or base pairs that are covered by binding of the protein Binding site: The sequence that a protein binds. The consensus sequence is the ideal binding site. Some positions require a specific nucleotide, others are less strict. 19  Modular protein organization Fig. 1.23 Brown genomes 5 Mol. Biology, Zlatanova, 2nd edition, Fig. 6.14 Some proteins contain multiple domains of different functions. RNA-binding proteins recognize a variety of folded structures, not just sites in the grooves as in DNA: Larger repertoire of RNA-binding protein motifs RNA protein modules recognize only a few bases, even only one, to achieve high specificity Proteins interact to form larger complexes Alberts, 7th ed. Fig. 3-41 Proteins bring together different components Interacting partners are often parts of complexes with common biological roles A network of protein-binding interactions in the cells of Drosophila Fig. 3.82, Alberts, 7th ed. Teamwork in Protein-DNA Binding Cooperative binding Cooperative A protein that binds a strong site assists the binding of binding another protein to a weak site. Increases the overall stability of the protein-DNA complex. Cooperative Recruitment: Multiple proteins are required to recruit a third one to specific DNA sites. Enhances precision, ensuring that only specific sites receive the appropriate cofactors needed for further functions Allostery: Protein binding can change the conformation, enabling or preventing the recruitment of cofactors. Fine-tunes cofactor recruitment allowing precise regulation of gene expression. https://doi.org/10.1093/nar/gkt1112  Intrinsincally disordered proteins Contain unstructured regions: lack a detectable tertiary structure, flexible Contain a limited subset of amino acids called low-complexity domains 1. Binding to other proteins 2. Signaling: often rich in phosphorylation sites (e.g., CTD of RNA pol). 3. Structural role Alberts, 7th ed. Fig. 3-76 Alberts, 7th ed. Fig. 3-73 Biomolecular condensates: Multiple interactions keep molecules together Alberts, 7th ed. Fig. 3-77 https://www.nature.com/articles/s41580-022-00566-8 Macromolecular machine (ribosome) Biomolecular condensate (nucleolus) Membrane enclosed compartment (mitochondrion) Alberts, 7th ed.  Studying protein interactions During cell lysis, proteins from different compartments may interact in the test tube: no physiological relevance. Interactions should be verified by different methods, in vivo and in vitro Biorender  Studying protein interactions Proteins may bind on the same DNA and RNA molecule (Direct protein-protein interactions persist during RNase or DNase treatment) Biorender 28 Nucleotides are the monomers Nucleotide structure: of nucleic acids A pentose: five-carbon sugar defines if RNA or DNA A phosphate group Α heterocyclic base (heterocyclic: atoms of at least two different elements) Nucleosides contain a base and a sugar, they lack a phosphate group Additional roles: energy currency, hormonal response, 2‘-Desoxy-b- enzymatic cofactors, metabolic intermediates. H D-ribofuranose OH :RNA 6 4 7 3 5 1 5 8 2 4 2 6 3 9 1 Modified Figures 8-1 and 8-2, Lehninger Principles of Biochemistry, 8th edition 29 Nucleic acids are nucleotide polymers Nucleic acids are linear polymers of mononucleotides: 3΄-5΄phosphodiester bonds Polarity: A free phosphate group at the 5΄end and a free hydroxyl group at the 3΄ end Oligonucleotides: usually shorter than 50 nts 30  Double helix DNA model 1. Two nucleic acid chains that wind about a common axis. Antiparallel: in opposite directions. 2. The backbone is on the exterior, nitrogenous bases on the interior. 3. The nitrogenous bases lie perpendicular to the common axis: stacked on one another. 4. Diameter of 20 angstroms. The B- form double helix has a wide and a narrow groove, called "major" and "minor groove". 31  Base stacking stabilizes further the double helix structure Base stacking reduces the contact of bases with water molecules and involves van der Waals and dipole-dipole interactions between the neighbouring bases. Base stacking interactions contribute to the stability of the double helix. Base flip-out signals DNA damage 32  Polarity and base-pairing are important for enzymatic reactions in gene expression The two strands are antiparallel: they have opposite 5'-3' and 3'-5‘ orientations. The different chemical nature of each end defines which reactions are thermodynamically efficient and many enzymatic activities have a specific orientation. Polymerases: Οne way: the 5΄to Exonucleases: Different degrees of 3΄way specificity 33 Polarity and base-pairing are important for enzymatic reactions in gene expression The two strands are complementary: the sequence of one, complements the sequence of the antiparallel strand (A-T, G-C base pairs). Lewins, Genes XII, fig. 1.22 34 Applications of base pairing in gene expression  DNA can undergo reversible strand separation Denaturation: Unwinding and separation of complementary strands. Increasing temperature: The double helix is destabilized and strands become separated because negatively charged phosphate groups pull apart the two strands. 36  DNA can undergo reversible strand separation Hyperchromic effect: Stacked bases in duplex DNA absorb less UV light than unstacked in single-stranded DNA. Double stranded DNA absorbs 40% less UV light than single stranded. Melting point (Tm): The temperature at which 50% of double stranded DNA is changed to single-standard DNA. Depends on: 1. GC pairs proportion 2. Ion concentrations 3. Agents that destabilize hydrogen bonds 4. Extreme pH values  Alternative DNA conformations: A and Z-forms B-form: the most stable conformation / Standard conformation/reference structure. A-form: absence of water Z-form: 1. Left-handed 2. Reduced width - 12 bases per helical turn. 3. The backbone runs in a zig-zag line. 4. Sequence-dependent: alternating purine-pyrimidine repeats (CG, 5mC-G Repeats) constitute a Z-helix under high salt conditions. 38  References Nelson & Cox. Lehninger Principles of Biochemistry, 8th edition. W.H. Freeman, chapters 8 and 24 Cox, Doudna and O’Donnell, Molecular Biology Principles and Practice, WH Freeman, chapters 5 and 6 Molecular biology of the cell,7th edition, Alberts et al., Chapter 3 Lewins, Genes XII, Chapter 1 Zlatanova et al, Mol. Biology, 2nd edition, Chapter 6 Brown, Genomes 5, Chapter 1 39