Biochemistry/Molecular Biology Review Sheet

B 1 ± Protein Structure and Function A. General Properties of Proteins a. Function ± catalysis, regulators, transporters, contractile elements, defense, structural elements i. Most weight in structural elements (collagen, elastin, keratin, etc.) b. Characteristics i. Composed of 20 amino acids through peptide bonds ii. Fibrous proteins ± resistant to changes in pH, temperature, and salt concentration iii. Globular proteins ± sensible to environment iv. Conjugated proteins ± combined with other components B. Protein Organization a. Primary structure ± amino acid sequence of L-configured AA i. An understanding critical for 1. Structural determination 2. Elucidating mechanism of enzyme action 3. Molecular basis of genetic diseases b. Secondary structure ± folding of polypeptide chain into repeating patterns i. Helical structures ± hydrogen bonds parallel to axis of helix ii. structure ± hydrogen bonds perpendicular to peptide chain c. Tertiary structure ± three dimensional structure of protein i. Stabilized by hydrogen bonds, ionic bonds, and hydrophobic interactions ii. Restrictions in peptide chain folding due to steric, bond rotations, and electrostatic interactions iii. Folding mediated by chaperones d. Quaternary structure ± non covalent association of protein subunits i. Stabilized by hydrogen, ionic, and hydrophobic interactions e. Protein complexes C. Structure/Function Relationship a. A slight change in the structure of a protein can have a devastating effect on its function i. Ex. CFTR mutation in F508, sickle cell anemia B 2 & 3 ± Enzymes 1 and 2 A. Functions of Enzymes a. Increase rate of reaction b. Regulate overall activity of cell c. Bind substrate at active site i. Site directed mutagenesis can help identify amino acids at the active site B. Enzyme Catalysis a. Energy of activation for chemical reaction/energy of activation graph b. Thermodynamic considerations i.  ii. At equilibrium, ¨G° = 0, thus ¨G°=-2.3RTlogKeq

+ cooperativity n > 1 ± sigmoidal curve 3. E¶B then turns into EQ and Q is released D. reaction is exergonic (Keq> 1) 3. then Q released b. ± cooperativity n < 1 ± decrease in effectiveness g.  . E binds to A to form EA ii. Specificity b. Regulation of Enzyme Activity a. Enzyme derivitization ± phosphorylation (very rapid) e. Sequential ± one at a time i. reaction is endergonic (Keq< 1) c. Mechanisms of Enzyme Reactions a. No cooperativity n =1 2. Protein synthesis and degradation (slowest) b. No change in 1/Vmax. Covalent intermediates iii. but no change in 1/Km 2. Enzyme inhibition i. Temperature and pH play a crucial role in enzyme activity C. Enzyme kinetics: Michaelis-Menten Equation  i. EA then binds to B to form EAB iii. change in 1/Km 2. Activation of enzyme precursors (ex. Km = [S] at .1. General acid-base catalysis iv. Double displacement i. Trypsinogen to trypsin) c. Multi-substrate Reactions a. 2 bindings sites) i. Activators and inhibitors affect MM curve in left to right . Allosteric enzymes (catalytic and regulatory sites. Cooperativity ± subunit communications between proteins 1. When ¨G° < 0. I cannot add a lot of substrate to overcome your effect e. Alteration of enzyme subunit interaction ± protein kinase. Isozymes ± variants of the same enzyme d.5Vmax d. Competitive inhibitors compete with substrate at active site 1. Proximity and orientation ii. Functional groups c. If you are a non-competitive inhibitor. When ¨G° > 0. E binds to A ii. Non-competitive inhibitors bind to non-active site location 1. Keq = [P]/[S] 2. P is released from EPQ. Change in 1/Vmax. If you are a competitive inhibitor. Catalytic efficiency (most important) i. adenylate cyclase f. I can add a lot of substrate to overcome you so will still reach Vmax ii. EAB transformed to EPQ iv. EA then turns to EP and P is released iii. E¶ then binds to B iv. Distortion of substrate E.

5¶-3¶ DNA polymerization b. Inhibitors increase Km B 4 ± DNA Replication A. DNA ligase links the two strands together to complete elongation .i. DNA Polymerase III a. Elongation 1. unwinds the DNA helix 2. Termination B. It replaces DNA Pol III at end to finish job 4. DNA Polymerase I a. Main replicative enzyme b. Activators reduce Km ii. Unwinding of helix 1. Used for DNA replication and repair b. Causes a nick in one strand 3. 3¶-5¶ exonuclease activity (proof reading) c. Has a beta clamp allowing it to be processive 3. Introduction to DNA Replication a. Has all three general activities of DNA polymerases c. 5¶-3¶ exonuclease activity to remove primer d. Occurs in a bi-directional manner from origin of replication 2. Dna B. Lays down short RNA primer to begin replication iii. DNA replication is a semi-conservative model e. Primase (RNA polymerase) recruited to replication fork a. Single strand binding proteins (SSBs) bind to separate strands to prevent them from pairing up ii. AT. Elongation iii. Initiation i. Dna A binds to origin of replication to allow helix to melt or unwind b. Initiation 1. DNA is genetic material of most cells. a helicase. Elongation i. DNA replication can be divided into i. except RNA viruses b. Priming 1. Fidelity of replication is essential for genome integrity of daughter chromosomes d. Topoisomerase 1 removes the torsional stress from helicase activity a. Has 5¶-3¶ DNA polymerization and 3¶-5¶ exonuclease activity c. Type 1 topoisomerase which is also present in eukaryotes b. Replication in Prokaryotes a. Cannot initiate replication 2. Each region served by origin of replication is called a replicon ii.GC paring c. DNA polymerases general points a.

will have two catenated daughter molecules 1. DNA Polymerase 1. Does not have clamp thus it is not processive iv. Once replication is done. trapping the replication fork between the terminator sequences iii. DNA polymerase used in mitochondrial DNA replication c. Diseases Arising from DNA Replication Defects a. Topoisomerase IV is a type 2 topoisomerase b. Expansion leads to pathological disease b. Helicases not well defined ii. Replication in Eukaryotes a. Replication of lagging strand is incomplete which can lead to shortening of chromosome 1. Has a clamp (PCNA) v. DNA Polymerase 1. Telomerase consist of 1. Separated strands coated by single strand binding proteins called RPA iii. Has integrated primase activity 2. Use topoisomerase IV to undo catenation a. The main replicative enzyme 2.c. Sticky ends of replicated chromosomes are prevented from self-ligation or ligation to other chromosomes by the formation of a T loop D. Elongation i. Reverse transcriptase ± enzyme extending chromosome ends iii. DNA Polymerase then comes back to fill in gaps and ligase joins fragments vii. Telomere shortening and incidence of cancer i. Each sequence acts as a binding site for DNA binding protein TUS ii. Termination i. This is prevented by telomeric repeat sequences 2. Termination i. Dyskeratosis congenita . Cuts both strands of the helix. Use Fen-1 to remove primers vi. Classified as non-Mendelian ii. Has proof reading activity 3. Occurs at terminator sequence 1. Telomeres are made up of multiple copies of repeated elements placed at the end of chromosomes ii. then reseals the nick after decatenation C. An RNA template 2. Expansion of trinucleotide repeat sequences i. Initiation ± poorly understood b. Does not have proof reading activity so error prone 3. TUS allows Dna B to pass through one way but not the other. No eukaryotic polymerase with 5¶-3¶ exonuclease activity 1.

Shortening of telomeres leads to unprotected ends that can fuse with others resulting in genomic instability and increase of cancer B 5. Lactose made from glucose and galactose . RNA transcript is synthesized 5¶-3¶ iii. Control can be by induction (turned on when needed) or repression (turned off when not needed) 6. Transcription of operon produces a poly-cistronic mRNA 3. Structure of gene or transcription unit i. 1 RNA polymerase composed of four subunits (2 . Transcription start site is defined by promoter region ii. Operons 1. LAC Operon 1. Transcription a. Telomere attrition after repeated cell divisions 2. Mitochondria 1.1. Prokaryotes 1. Transcription stop site is defined by terminator sequence c. DNA topoisomerases aids in unwinding of double helix v. Each promoter is controlled by one or more control elements 5. General Mechanism i. RNA polymerase 1 (large rRNAs). Each structural gene in the operon has its own translation start signal to attract ribosomes ii. Eukaryotes 1. Allows for coordinated expression 4. rRNA (3-4). Only transcribe one strand b. Have their own RNA polymerase B. mRNA (1000s) d. RNA polymerase II (mRNAs). Increased cancer risk with aging 1. Types of RNA i. Induction by lactose a. No primer iv. Mutation in RNA component of telomerase leading to abnormal shortening of telomeres ii. 1 ¶) ii. RNA polymerase reads template strand from 3¶-5¶ ii. Transcription machinery i. Different factors help each polymerase recognize promoter and initiate transcription iii.6 ± Transcription and Control of Transcription A. RNA polymerase III (tRNAs and small rRNA) 2. tRNA (~50). Prokaryotes i. Regulation of Transcription a. Organized clusters of genes under control of a single regulatory signal or promoter 2. 1 .

Structure and function of a promoter for a typical eukaryotic gene 1. lactose binds to repressor. need to synthesize more 4. breaks down lactose to glucose and galactose e. Trp operon encodes many enzymes needed to synthesize the amino acid tryptophan 3. repressor binds to operator and blocks transcription again f. A protein product from the operon. Binding may be controlled by environmental conditions ii. Contribute to regulation b. If glucose exhausted. trp repressor protein cannot bind to operator site so trp operon is transcribed b. When no lactose.b. If galactose and glucose present. How binding of transcription factor activate transcription . The transcribed gene is composed of exons and introns 2. causing a change in its shape which prevents it from binding to the operator d. When lactose is exhausted. The initiator sequence overlaps with the transcription start site (TATA box) 30 bases before the start site or binding site for transcription factors that associate with RNA polymerase II b. cAMP is low i. TRP Operon 1. Repressed by tryptophan 2. When lactose present. Promoter a. operon is not very active because glucose represses it since energy from glucose is more efficient b. Enhancer and silencer elements can be close or far away from start site and can be before or after transcription units and even between introns 3. cAMP can start this cycle because it is high iii. Mammalian cells i. lac repressor protein (made from gene adjacent to lac operon) binds to an ³operator´ sequence in the promoter of the lac operon and prevents transcription c. Function of enhancer/silencer elements a. When glucose is present. When tryptophan is low. cAMP is must bind to CAP to change its shape to bind to lac promoter ii. Repression by glucose a. In absence of tryptophan. CAP helps recruit RNA polymerase to start transcription iii. The lac mRNA has short half-life (3 min) 2. beta-galactosidase. Each one is a binding site for a specific transcription factor i.

General characteristics of hormones a. N-terminus histone tails extend beyond the DNA and interact with other factors i. Eukaryotic DNA packed in chromatin a. Some transcriptional activator proteins bind directly to basal transcription factors to help recruit RNA polymerase II to the promoter iii. Distinction between cell surface and nuclear receptors for hormones a. Partially conserved hormone binding domain on the Cterminal end b. progestins. Examples 1. DNA wrapped around histone octamer c. Hormones that bind to receptors on cell surface (insulin. catecholamines) activate protein kinases and/or phosphatases i. Basic unit is nucleosome i. Enter cell. androgen. retinoic acid 3. They are intracellular messengers (go into the cell) b. cortisol. Amino acid derivatives ± thyroid hormones 3. aldosterone. Steroid-related ± vitamin D. Coactivators alter chromatin to make promoter more accessible b. They in turn release 2nd messengers b. estrogen. binds to receptor in cytoplasm or nucleus b. Structure of nuclear hormone receptor proteins a. Other hormones bind to intracellular receptors i. progesterone 2. These interactions control chromatin openness iv. Steroids ± glucocorticoids. testosterone. Coactivators recruit RNA polymerase II and its basal transcription factors 2. estradiol. Activate pre-existing pathways 2.1. Response pathway of steroid hormones a. Highly conserved DNA binding domain in the middle 4. mineralocorticoids. Composed of 2 copies each of 4 different histones that are spaced about 200 base pairs apart on DNA b. Transcriptional activator proteins (once they bind to enhancer) help recruit coactivators a. Role of chromatin structure in regulation of transcription 1. Hormone-activated receptor forms dimers and binds once specific type of enhancer element c. DNA bound receptor recruits coactivators . Steroid and nuclear hormone receptors (examples of transcriptional activator proteins) 1.

Position of transcription initiation . CREB binds to a specific enhancer element (CRE) vi. rRNA. PKA phosphorylates many proteins to change their activities including cAMP Response Element Binding protein (CREB) 4. 7SL RNA. Acute promyelocytic leukemia i. XIST (involved in X chromosome inactivation). Cancer a. Retroviruses can reverse transcribe RNA to DNA ii. Types of RNA in mammalian cell i. use antagonist to suppress it c. Spinal and bulbar muscular atrophy (mutation in androgen receptor) B 7 ± RNA Processing A. Clinical Correlations 1. Processing events determine what mature mRNA looks like b.v. mitochondrial RNAs. tRNA. Breast i. mRNA. Tumor growth depends on estrogen so block it using estradiol or use tamoxifen which prevents coactivators from binding b. Chromosomal translocation creates oncogene 2. Genetic diseases a. Hormone resistance due to mutation of nuclear receptor gene b. siRNA 1. Binding of hormone to cell receptor causes release of second messenger like cAMP 2. Creutzfeldt-Jakob¶s disease 2. Here a misfolded protein induces the misfolding of other proteins b. Overview of mRNA Processing a. These are inhibitory RNAs that are produced from double stranded precursors 2. Leads to mad cow disease or in humans. cAMP binds and activates PKA 3. snRNA (processing prerRNA). The central dogma of DNA to RNA to Protein is not set in stone i. siRNA or RNAi ii. Chromosomal translocation creating oncogene d. Introduction a. telomerase RNA. Tumor growth depends on androgen. snRNA (for splicing). Prostate i. Prions (proteins) can act as infectious agents 1. Can destroy target mRNA or repress translation B. Acute myeloid leukemia i. Regulation of transcription by cAMP via CREB 1.

Exons can be coding or non-coding Events Following RNA Processing a. mRNA exported i. Aging is accelerated 2. Alternative splicing allows exons to be joined in different ways e. Can be done by deletion of exons. mRNA translation d. Cleavage of 5¶ splice site ii. mutation of splicing signal. translation. mRNA localization i. Adding a Poly A tail for mRNA export. Position of transcription termination and polyadenylation i. i. If conserved sequence of intron is mutated. Disruption of splicing i. mRNA destruction Diseases Related to mRNA Processing Defects a. Exons are short (~200 base pairs) c. F. Activation of cryptic splice site leading to frameshift mutation iv. D. and stability c. Occurs in three steps i. Cystic Fibrosis is an example of disruption of splicing 1. splicing may be affect by not removing that intron i. helps regulate where certain proteins are made c. Removing introns and joining exons together ii. Introns can be inside or outside coding region iii. E. Ligation of 5¶ splice site to 3¶ splice site b. or activation of cryptic splice signals ii. Cap structure added immediately following transcription that is required for mRNA export. and stability d. Changes in both coding and non-coding can have a huge effect b. export is prevented if not all the steps are completed b. Will prevent mRNA export and cause pre-mRNA degradation iii. Hutchison-Gilford progeria 1. Introns can be short or long b. Most conserved splicing sequences are found in the intron c. Mutation in cryptic splice site in the lamin A/C gene . splicing and polyadenylation sites Mechanism of Splicing a. Splicing i. Attack of invariant branch point A to the 5¶ G of the intron yielding a lariat iii. One way it can still be removed if there is an alternative (cryptic) splice site nearby Characteristics of Introns and Exons a. Some can splice autocatalytically but most cells require snRNPs to mediate splicing in a large complex called spliceosome e. Can make different mRNAs from one gene due to alternative transcription sites. translation.C. Exon sequences flanking the intron show weak conservation d.

Altered mRNA decay i. . allowing it to live longer and contributing to the proliferation B 8 ± Translation A.c. Chromosomal translocation causing deregulation of mRNA decay. Burkitt¶s lymphoma 1.

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