Flashcards in CMB self-study Deck (133):
Prokaryotes vs Eukaryotes
Prokaryotes: no membrane enclosed organelles, chromatin in nucleoid Eukaryotes: membrane bound organelles and vesicles, nuclear membrane
Adenine (A) Guanine (G)
Cytosine (C) Thymine (T) in DNA only Uracil in RNA only
A bonds with T or U (2 bonds) and C bonds with G (3 bonds)
Purines and pyrimidine bases + pentose (deoxyribose or ribose)
Phosphorylated nucleosides (5' -OH of ribose or deoxyribose is phosphorylated)
Short polymers of nucleotides (up to 30)
Role of Mg2+ and other cations
Shield phosphate groups from electrostatic intrastrand repulsion (balance the negative charges)
Why is circular DNA an advantage?
May provide protection from degredation by exonucleases
Includes substrate-binding site and catalytic site
Contains catalytic residues which act on the substrate
-ase usually means
Conformation primarily found in cells
Form taken by DNA-RNA hybrid during transcription
Occurs within DNA sequences that control gene transcription
Causes of denaturation
Extreme pH, extreme ionic strength, high temp
Type I isomerases
Break only one DNA strand and allow it to rotate about the other to relieve supercoil. Also reseal (ligate) break
Type II isomerases
Act as ATPases-Using ATP, break both DNA strands, relax supercoil, reseal DNA
What are gyrases?
Topoisomerases which relieve supercoiling from unwinding of DNA. Prevent supercoiling that would be induced by unwinding DNA in DNA synthesis
Antibiotic used for antibotic resistant UTIs. Inhibit gyrases and interferes with breaking and rejoining of DNA
Antibiotic which blocks the binding of ATP (which blocks type II isomerases)
Regions of DNA which interchain hydrogen bonds are broken and intrachain bonds form. Function in the control of replication and transcription
Triplex DNA functions and bonding
Triple stranded DNA. Usually in regions with string of purine bases. Functions in transcription control, initiation and termination of replication by enhancing stability of chromosome ends called telomeres. Forms hydrogen bonds with major groove of B-DNA with Hoogsteen pairing.
What is triple-stranded DNA associated with?
Hereditary persistance of fetal hemoglobin. Mutation prevents the triple stranded DNA from forming and fetal hemoglobin to continues to be transcribed
Occurs on different faces of DNA than Watson and Crick pairing
Forms in immunoglobin genes that undergo recombination. Responsible for antibody diversity. Also present at the telomeres.
Histones (types, function)
Eight proteins (2 each of H2A, H2B, H3, H4) combine to form a histone disk octamer around which DNA supercoils. Interact with highly acidic phosphate residues of DNA in minor groove. H1 is not part of the octamer and functions to stabilize the DNA around the octamer. Histone genes have NO INTRONS.
1 _ turns of DNA with the histone disk but no H1
2 full turns of DNA with the histone disk + H1
Linear arrays of nucleosomes and chromatosomes
Heterochromatin vs Euchromatin
Heterochromatin is tightly packed and euchromatin is loose-more accessible and transcriptionally active
What do prokaryotes have instead of histones?
Prokaryote chromasome structure
One circular double stranded supercoil chromasome
Bacterial chromosomes are compacted into ________ by interaction with HU proteins, cations, polyamines, RNA, and other nonhistone proteins
Each DNA strand is self-complimentary within the inverted region that contains the symmetry elements
Identical base pairs equidistant from center of symmetry within DNA segments
Particular sequence repeated
Size of human genome
Function of primer
Provides free -OH group to which nucleotides can be added
What is released as nucleotides are added to growing DNA strand?
Pyrophosphate (PPi) is released whenever phosphodiester bonds are formed
Which end of the strand are nucleotides added to?
How are primers removed?
Exonuclease activity removes primer nucleotides in 5' to 3' direction
Unwind the DNA strands to allow them to separate
Single-stranded binding proteins (SSB)
Bind to DNA to keep the two strands from annealing
Types of prokaryotic DNA polymerases and functions
3 types: Pol I, Pol II, Pol III. All have 3'-->5' exonuclease proofreading activity. Pol I (intermediate processivity) synthesizes lagging strand and only one capable of primer removal (5'-->3' exonuclease activity). Pol II (low processivity) involved in DNA repair. Pol III (high processivity) synthesizes leading strand.
Donut shaped protein complex which encircles DNA strands. Prevents Pol III from detaching from the template until replication complete. Reason why Pol III has high processivity.
Ability of the enzyme to remain on its substrate during synthesis
Includes primosome, SSB proteins, Pol I and Pol III, other molecules needed for DNA replication
Primases, ligases, helicases, and other proteins that bind to the origin of replication and are necessary for synthesizing the primer
DNA segments between the origins of replication
Differences between prokaryote and eukaryote DNA synthesis
Eukaryotic DNA is much larger, packed into chromatin, and have lower rates of replication fork movement than in prokaryotes
Cell cycle order and events
M, G1, S, G2. Mitosis is where cell division occurs. In G1 cell increases in size and makes extra proteins, organelles--G1 checkpoint makes sure cell ready for S phase. In S DNA replication occurs. In G2 rapid cell growth and protein synthesis--G2 checkpoint makes sure cell ready to divide. Also can leave G1 and go into G0 resting phase when the cell has stopped dividing.
Cyclin-dependent kinases (CDK)
Controls the cell cycle by initiating cell division and DNA replication
Protein substrates of CDKs and activate them
Types of eukaryotic DNA polymerases and functions
2 types: _ and _. Polymerase _ (low processivity) synthesizes lagging strand, functions as a primase, and disassembles nucleosome prior to DNA replication. Polymerase _ (high processivity) synthesizes the leading strand, serves as 3'-->5' exonuclease proofreader.
Proliferating cell nuclear antigen (PCNA)
Serves as a clamp to kee the polymerase _ enzyme from dissociating. ::homologous to the sliding clamp in prokaryotes::
Replication protein A (RPA)
Bind to DNA to keep the two strands from annealing ::homologous to the SSB proteins in prokaryotes::
When does synthesis of histones occur?
During S phase, simultaneous to DNA replication
Enzymes that use RNA as a template for DNA synthesis (RNA dependent DNA polymerase)
Telomerase (definition, structure, and function)
Reverse transcriptase that replicates the telomeres. Contains RNA as part of its structure so functions as a template and an enzyme during replication. No proofreading so most error-prone of the DNA polymerases.
Genomes are RNA. During viral infection RNA is copied into DNA and transcribed to produce more viruses.
Inhibits reverse transcriptases. Structural analog of dT, converted to triphosphate and incorporated into viral genome in place of dTTP (AZT competes and has a higher binding affinity than dTTP). Once incorporated into viral DNA, AZT causes premature termination of viral DNA synthesis because it lacks the 3' -OH site needed for the addition of nucleotides.
3 types of recombination
1. Homologous: occurs between homologous DNA on the same chromosome during prophase of meiosis. 2: Conservative site-specific recombination: insertion of bacteriophage DNA into bacterial genome. Homology is not required. Double stranded DNA is inserted at specific sites. 3: Transposition: Jumping genes which move from one site to another within a chromosome. May activate or inactivate gene if inserted into coding sequence. This can make bacteria antibiotic resistant by carrying transposons on plasmids (which can replicate independent of organism's DNA).
Types of RNA modified bases:
1. pseudouridine 2. dihydrouridine 3. methylated bases: methylguanosine, dimethylguanosine, methylinosine 4. ribothymidine
Single stranded RNA makes these by base pairing with itself
mRNA in prokaryotes
Polycistronic: can code for more than one protein
Segment of DNA that contains all of the information for production of a single polypeptide
mRNA in eukaryotes
Mostly monocistronic: can only code for one polypeptide
Shortest of the RNAs (mins to several hours). Exceptions: mRNA in unfertilized eggs (dormant until fertilization) and mRNA that codes for hemoglobin in reticulocytes (very stable).
Post-translational modifications to mRNA
1. Inverted methylated base 5' cap: inverted from 5' phosphate to 5' phosphate linkage with 1st nucleotide (often methylated purine) instead of usual 3',5' phosphodiester bond. 2. Poly A tail: 20-200 adenine nucleotides, length of tail determines stability. Histone mRNA lacks poly A tail.
Untranslated leader sequence following the 5' cap in prokaryotic mRNA. There is also a nontranslated sequence following the stop codon.
Untranslated leader sequence following the 5' cap in eukaryotic mRNA. There is also a nontranslated sequence following the stop codon.
UAA, UAG, UGA
Amounts of each type of RNA in cells
80% rRNA, 15% tRNA, 1% mRNA, the rest is other RNA
Cloverleaf with 2 active sites: 1. Acceptor stem 3' -OH terminal CCA where amino acids attach 2. Anticodon loop which recognizes codons on the mRNA.
tRNA activation and functions
Needs to be activated (aminoacylated) by aminoacyl-tRNA synthetase. Has 2 functions: 1. Activate the amino acids for protein synthesis which occurs on the ribosome by binding to the acceptor stem 2. Recognize codons to ensure correct amino acid incorporated into the polypeptide chain using the anticodon loop
Two components: large and small particles which are named according to sedimentation coefficient during centrifugation calculated in Svedberg units.
Small nuclear RNA (snRNA)
Recognizes introns on mRNA participating in splicing
Small cytoplasmic RNA (scRNA)
Involved in selection of proteins for export where it serves as signal recognition particle
Mitochondrial RNA (mtRNA)
Include mRNA, tRNA, and rRNA transcribed from mitochondrial DNA. Only one tRNA for each amino acid.
Ribonucleoprotein particles (RNP)
Small RNA/protein molecules which function in RNA processing, splicing, transport, control of translation, and protein recognition particles that target proteins for export. The RNA component of the RNP is a ribozyme.
Ribonuclease P (RNase P)
True enzyme which cleaves pre-tRNA to generate mature 5' terminus of tRNA
Control cell's phenotype by shutting down developmental genes or altering levels of expression. Control cellular differentiation.
In what direction is DNA read? What direction are DNA and RNA transcribed?
DNA is read in the 3'-->5' direction. DNA and RNA are transcribed in the 5'-->3' direction
Found at the -10 region with the sequence TATAAT. Recognized by the RNA polymerase which attaches before the beginning of transcription.
Differences between DNA and RNA polymerases
RNA polymerases requires DNA template but no primers. RNA polymerases have no proofreading ability.
Structure of RNA polymerase
Holozyme consists of all 6 subunits: two _, _, _', _ (omega), _ (sigma). Without _ called the core enzyme.
RNA polymerase _ subunit function
Recognizes the promoter and then is released as the RNA polymerase begins to synthesize the RNA
Types of RNA polymerases and inhibitor sensitivity
3 nuclear (I,II,III) and 1 mitochondrial type. Distinguished by sensitivity to amanitin (mushroom poison)/rifampicin (antibiotic).
RNA polymerase I
Produces rRNA, insensitive to _-amanitin. Synthesizes a single transcript of the rRNA subunits at the same time to form pre-RNA (this ensures equal molar amounts of each subunit).
RNA polymerase II
Produces mRNA, very sensitive to _-amanitin. Present in the nucleus.
RNA polymerase III
Produces tRNA, sensitive to _-amanitin. Present in the nucleus.
Mitochondrial RNA polymerase
Produces all kinds of RNA, insensitive to _-amanitin but sensitive to rifampicin
Rifampicin as a drug
Treats Mycobacterium tuberculosis by inhibiting prokaryotic RNA polymerase by binding to the _ subunit.
rRNA gene location
Nucleolar organizer region of the nucleolus. Several hundred copies of each rRNA gene separated by spacer sequences.
3 general modifications of the RNA primary transcript
1. Removal of external (terminal sequences) and internal (spacer sequence) nucleotides by ribonucleases 2. Base modification 3. Addition of nucleotides
Post-translational modifications to tRNA
1. 5' end removed by ribonuclease P (ribozyme) 2. 3' end removed and terminal CCA is synthesized 3. Nucleotide bases modified (tRNAs contain the most modified bases of all nucleic acids)
Splicing (proteins involved, details, important sequences)
Small nuclear ribonucleoproteins called snRNPs (snurps) remove introns from pre-RNA. Break RNA at the 5' end of intron and join exons. All introns begin with GU (donor site) and end with AG (acceptor site) but not all GU or AG sequences are splice sites. U1RNA, U2RNA, and snurps discriminate the splice sites.
about 5 days
What makes the genetic code degenerate?
Amino acids have more than one codon except for Met and Trp
Which amino acids have only one codon
Met (methionine) and Trp (tryptophan)
Why no wobble effect with G at 3' position?
Makes sure there is no ambiguity coding for the stop codon, Trp, or Met
Aminoacyl-tRNA synthetase functions
Catalyze two reactions and has proofreading ability 1. Activation of amino acid with ATP forming the aminoacyl group 2. Transfer of the aminoacyl group to the tRNA
Methionyl-tRNAs in prokaryotes
1. Initiator tRNA: fMet-tRNA has formylated Met group and initiates protein synthesis 2. Carries Met for other positions in the protein (not formylated)
Methionyl-tRNAs in eukaryotes
No formylation of Met so all methionyl tRNAs are identical
Steps of protein synthesis
1. Initiating fMet-tRNA (prokaryotes) moved to P (peptidyl) site of ribosome small subunit 2. Large ribosome subunit joins complex 3. Peptidyltransferase transfers the initiating fMet-tRNA (prokaryotes) to E (exit) site and 2nd amino acid in sequence placed in the A (aminoacyl) site on ribosome 4. Peptide bond forms between amino acids 5. Elongation occurs until stop codon reaches the A site (no aminoacyl-tRNA binds with these codons).
Attachments on an amino acid
Carboxylic group (-COOH), Amino group (-NH2), Hydrogen (-H), specific side chain (-R)
What isomer of amino acids are in humans?
L amino acids (usually rotate polarized light to the left but not always)
Characteristics of peptide bond
Characteristics of a double bond so no rotation about the C-N, but _ carbons can rotate
Peptides vs proteins
Peptides are < 50 amino acids, proteins are > 50 amino acids
Disulfide bond role
Stabilize protein structure and involved in oxido-reductive reactions
Most important agent that fights reactive oxygen species. Composed of Glu-Cys-Gly. When membrane proteins get oxidized, glutathione reduces them.
pH=pK + log (A-/HA) In bicarbonate buffer system equation is pH=pK + log (HCO3-/H2CO3) Increase in HCO3- causes pH to rise.
Primary protein structure
Order of amino acid residues
Secondary protein structure
Local 3D folding of the chain
Tertiary protein structure
3D structure of the protein which folds into domains. Hydrophobic side chains are inside away from water and hydrophilic side chains are on the outside and interact with water. Structure stabilized by noncovalent electrostatic, hydrophobic, hydrogen, and disulfide bonds.
Quarternary protein structure
non-covalent association of discrete polypeptide subunits into a multi-subunit protein. Subunits are noncovalently attached. ::not all proteins have a quarternary structure::
Protein secondary structure types
_-helix, _-pleated sheet, _-turn, random coil. Side chains do not participate in formation of secondary structures.
L-amino acids form right handed _-helices. Kept together by H-bonds between carbonyl (C=O) groups and imino (N-H) groups directly above or below each other (~every 4th amino acid)
Two polypeptide chains are aligned paralell or antiparalell. H-bonds between carbonyl (C=O) groups and imino (N-H) groups between adjacent chains.
Reverse the direction of the polypeptide chain allowing the chain to form compact globular proteins
Anything that doesn't fit in the other secondary structure categories. Less ordered structure. Functional and found in binding sites or active centers of proteins.
Advantages of quarternary structure
Stability, genetically economical (in homomultimers only one gene needed to code for a complex protein), efficiency (active sites brought in close proximity and substrate channeled from one subunit to the other without diffusion and/or transport), cooperativity
Final conformation of the protein which is the most stable (disulfide bonds form to help stabilize)
Belong to Heat Shock Protein family. Synthesis increases at high temps. Required for refolding proteins as they cross membranes, assembling proteins with multiple polypeptide chains (quarternary structure), and facilitating protein transport into the mitochondria and endoplasmic reticulum
4 types of bonds that stabilize proteins
1. Disulfide bonds: covalent linkages between -SH groups in cysteines to produce cystine (called a sulfide bridge) 2. Hydrophobic interactions: hydrophobic groups on inside of protein 3. Hydrogen bonds: between amino acid side chains and stabilize tertiary structure 4. Ionic (electrostatic interactions): salt bridges and can be repulsive or attractive--formed between positively and negatively charged side chains
Disrupts all but the primary structure. Occurs due to strong acids, strong bases, heating above 60 degrees C, or by adding denaturants like urea or detergents like SDS or guanidine. In cell leads to enzymatic removal of protein.
Proteins fold in abnormal way and form aggregate long, fibrillar protein assemblies called amyloids