Test 1 Flashcards
Endosymbiont model
- Bacterial endosymbiont engulfed by archael membrane fusions
- Archael membrane fusion protrusions expanded to fully engulf
(precursor to aerobic eukaryote)
E3 model / common features of both models
Entangle, engulf, endogenize
An ancient anaerobic archael cell, an ancient aerobic bacterium, and over evolutionary time, a symbiotic relationship between the two
Evidence of endosymbiant hypothesis
- Mitochondria / chloroplasts still have remnants of their own genomes and their genetic systems resemble that of modern-day prokaryotes
- Mitochondria and chloroplasts have kept some of their own protein and DNA synthesis components and these also resemble prokaryotes
- Membranes in mitochondria and chloroplasts often similar to those in prokaryotes and appear to have been derived from engulfed bacterial ancestor
Info flow in the cell: the central dogma
DNA - transcription - RNA - translation - PROTEIN
Genome
All DNA in the cell (includes mitochondrial and chloroplast DNA)
- doesn’t change
Transcriptome
All RNA in the cell at a particular point in time
- changes constantly
Proteome
Protein in a cell at a point in time
- changing constantly
Interactome
Set of protein-protein interactions happening in a cell at a point in time
Metabolome
Set of metabolites found in a cell at a point in time (smaller than proteins ex. sugars, ATP, vitamins)
Phenome
Whole collection of -omes together with some characteristic (way to connect -omes with physical traits)
What are nucleic acids
The genetic material in a cell: organism’s blueprints
- DNA, RNA
3 parts of a nucleotide
- Pentose sugar
- Nitrogenous base
- attached to 1’C
- gives nucleotide its identity - Phosphate group (1, 2, or 3)
- gives DNA its overall negative charge
Nucleoside, deoxyadenosine
Just a base and a sugar, no phosphate
(Nucleoside monophosphate, diphosphate, triphosphate)
Deoxyribose sugar instead of ribose sugar with adenosine nitrogenous base
Nucleic acid chain synthesis and linkages
DNA is synthesized from deoxyribonucleic triphosphates, otherwise known as dNTPs
RNA is synthesized from ribonucleoside triphosphates or NTPs
Nucleotides are linked by phosphodiester bonds
Three forces that keep DNA strands together
H bonds, hydrophobic interactions, van der Waals attractions (temporary dipole interactions becomes important in great numbers such as crowded DNA arrangements)
DNA structure
Energetically favourable conformation
Protein can recognize and bind to sequences in major and minor grooves
Two ends of DNA strands
5’ Phosphate group
3’ OH group
Protein structure: hierarchal organization
Primary (sequence)
Secondary (local folding)
Tertiary (long-range folding)
Quarternary (multimeric organization)
Multi protein complexes
Amino acid structure / major categories of amino acids
R group determines type of amino acid
R group, amino group, alpha carbon, carboxyl group
Basic, acidic, nonpolar, uncharged polar
Roles of amino acid types
POLAR
Negatively/positively charged: enzymatic function, shape
Uncharged: on the outside of proteins to interact with water (cell is full of water)
NONPOLAR
Often form inner core of proteins since they are hydrophobic, associated with lipid bilayer in proteins
Cysteine: disulphide bonds
Oxidizing conditions can create bond between cysteines in two polypeptide chains (interchain) or within a single peptide (intradisulfide bond)
- very strong covalent bond
- important for proteins undergoing chemical/mechanical stress
- in cytosol, less needs for these bonds as reducing conditions are favoured
Peptide bond formation
Reaction between carboxyl group of one amino acid and amino group of another amino acid
Hydrolysis releases water (peptide bond formed by a condensation reaction)
Primary protein structure
Amino acid has a carbonyl, no longer carboxyl, peptide bond to N of adjacent amino acid
N terminus and C terminus
NCC backbone
Once an amino acid has been joined by a peptide bond, it is called a …
Residue, due to the effect of the loss of the water molecule
Alpha helix structure
R groups not involved
Bonds form 4 residues apart between carbonyl oxygen and amide hydrogen parallel to axis of helix
3 amino acids per turn, begin to see helix after 2 bonds form
Alpha helix vs DNA double helix
ALPHA HELIX
- single stranded
- R-groups face out
DNA DOUBLE HELIX
- usually double-stranded
- bases face out
Both have polarity, but of different types
Beta sheet
H-bonding between carbonyl oxygen and amide hydrogen of amino acid in a neighbouring polypeptide strand
R groups not involved, but alternately point up and down
Typically contain 4-5 strands but can have 10+
Two types of beta sheets
Anti-parallel
Parallel
- longer due to extra looping to connect strands
H-bonding in secondary structures summary
Which atoms H-bond?
Carbonyl oxygen, amide hydrogen in peptide backbone
Alpha helices
4 amino acids apart within the same segment of the polypeptide chain
Beta sheet
Between amino acids in different segments or strands of polypeptide chain
Coiled coil
Two helices wrap around each other to minimize exposure of hydrophobic amino acid side chains to aqueous environment (hydrophobic R groups get pushed into the middle)
Strong structure: found in alpha-keratin of skin, hair, motor proteins
Amphipathic (type of alpha helix)
Means it has hydrophobic and hydrophilic areas, coiled coil is an example of an amphipathic alpha helix
Tertiary structure
3D overall structure of a protein held together by:
- hydrophobic interactions
- non-convalent bonds
- covalent disulfide bonds (“atomic staple” which really helps hold together tertiary structure)
Proteins generally fold into the most energetically favourable conformation
Proteins fold into the shape dictated by …
Their amino acid sequences.
But chaperone proteins help make the process more efficient and reliable in living cells
Protein domains
Specialized for different functions
Portion of a protein that has its own tertiary structure, often functioning in semi-independent manner
Connected by intrinsically disordered sequences
(Whole protein and all domains still made up of one continuous polypeptide)
Protein families
- similar amino acid and tertiary sequences
- members have evolved to have different functions
- most proteins belong to families with similar structural domains
Quarternary structure
Only in proteins with more than one polypeptide chain, held together by covalent bonds
- each subunit in separate polypeptide
Ex. Hemoglobin has 2a and 2b subunit
Multi protein complexes and molecular machines
- Can be many identical subunits (ex. actin filaments)
- Mixture of different proteins and DNA/RNA (ex. viruses and ribosomes)
- Very dynamic assemblies of proteins to form molecular machines (ex. for DNA replication initiation or transcription)
Scaffold proteins
Get interacting proteins close together so they can interact more quickly
How are proteins studied
- purify protein (separate from other cell material)
- determine amino acid sequence (can be by mass spectrometry)
- discover precise 3D structure (NMR, crystallography, AI)
Proteomics
Large scale study of proteins, researchers use a range of approaches to collect data on the set of proteins in a sample including
- identity and structure
- protein-protein interactions
- adbundance and turnover
- location in cell or tissue
1 kb
1000 base pairs (one kilo base pair)
Bacterial genome shape
Circular
Organelles genomes
Circular
- support for endosymbiant theory
Much smaller than bacterial genome
- because some genetic info has revolved to be in nuclear genome instead
Human genome
- 3 billion base pairs (haploid set)
- 6 billion base pairs per cell (one set from each parent)
- 20000 protein-coding genes spread across 23 pairs of chromosomes per cell
- 2n aka diploid
Genome size is correlated with number of genes / organism complexity
False, not always
Repeated sequences: mobile genetic element (human genome)
Less than 14 bp repeated over and over
Half of human genome
Most no longer move, evolutionary fossil
Unique sequences (human genome)
Nonrepetitive DNA that is neither introns nor exons (things like promoter sequences, regulatory sequences), introns, exons
- other half of human genome together with repeated sequences
Prokaryotic DNA packaging
DNA is condensed through folding and twisting about 1000 times and complexed with proteins to form prokaryotic nucleoid, where condensed DNA and non-histone proteins are found (not membrane-bound)
Chromosomes in prokaryotes vs eukaryotes
Prokaryotes: circular
Eukaryotes: linear
Eukaryotic DNA packaging
Chromosomes
- 23 pairs
- each chromosome contains a single long linear DNA molecule called chromatin
- DNA must remain accessible and tightly packed
Chromatin
- single, long, linear DNA molecule which folds into a chromosome
- dynamic, changes throughout life of a cell
Chromatin levels of organization
- DNA double helix
- Beads on a string
- Chromatin fiber of packed nucleosomes
- Fiber folded into loops
- Entire mitotic chromosome (10000 fold shorter than extended length)
Nucleosome (beads in the beads on a string)
- Core particle
- Double stranded DNA wrapped around
- Linker DNA to link to other core particles
DNA wraps twice around each nucleosome
Each has 200 nucleotides
Histones
Small proteins rich in arginine and lysine
Positive charge so DNA doesn’t repel itself
Four core histone proteins with N-terminal tail which can be modified and one linker histone
Packaging of nucleosomes
Sequence-specific clamp proteins bind to DNA and cohesins form chromatin loops
As cell enters mitosis, condensins replace cohesins to form double loops of chromatin for more compaction
Result is metaphase chromosome
Heterochromatin vs Euchromatin
Heterochromtin
- highly condensed
- regions of interphase chromosomes where gene expression is suppressed
Euchromatin
- non-condensed chromatin
- areas where genes tend to be expressed
Fluid movement between states at any given time
DNA replication
Semiconservative
5’ to 3’
- nucleotides added on 3’ end
Starts at specific origins of replication in a chromosome
DNA replication procedure
- Separate strands
- Synthesized new strands
- Proofread new strands
6 Ingredients for DNA synthesis
- original of replication
- primers
- dNTPs (nucleotides)
- ATP
- DNA polymerase
- accessory proteins
How many origins of replication in bacterial and linear chromosomes
Bacterial: 1
Linear: 2
Initiator proteins
Recognize and bind to origin of replication
Helicase
Unwinds DNA double helix
- predominant type moves 5’ to 3’ along the lagging strand template
- requires ATP
SSBPs
Keep helix separated and untangled (prevents reannealing)
RNA primers
Made by primase
Makes RNA primer for DNA polymerase to start synthesizing DNA
DNA polymerase
Adds nucleotides onto 3’ OH
Sliding clamps
Hold polymerase onto DNA so it doesn’t fall off
Allows it to proceed more quickly
Needs ATP and clamp loader to be loaded onto template strand
DNA ligase
- Seals nicks in 5’ to 3’ fragments (between Okazaki fragments)
- Removes RNA primer sequences and replaces the gaps with DNA
Origin of replication contains
AT rich sequence (only 2 H bonds so it is easier to break)
Binding of initiator proteins requires energy to…
Regulate replication
In order to begin, DNA polymerase requires…
- Bound primer
- Template
- 3’ OH to add nucleotide onto (provided by primer)
Primase main purpose and direction
Synthesize RNA primer in 3’ to 5’ direction along template strand
How does DNA polymerase add new nucleotides
Clips off 2 phosphate groups for energy and to allow formation of phosphodiester bonds
Nucleases
General term for enzyme that digests nucleic acid
Replisome
Example of a molecular machine used in bacterial DNA replication
Topoisomerases
Create transient single-strand break to alleviate tension and strain in DNA as it is unwound by helicase
Which strand has a gap that remains unreplicated at the end? Why is this a potential problem?
Lagging strand (no 3’ OH gap)
Loss of genetic information over time
Telomerase
Enzyme that adds nucleotides to telomeres without needing a primer
Telomere replication generates
G-rich ends
Telomeres are abundant in…
Stem and germ cells, not in somatic cells
Telomeres and cancer
Cancer cells produce high levels of telomerase, with high replication levels contributing to instability of cancer cells
DNA or RNA polymerase more accurate?
DNA polymerase
Two mechanisms of DNA proofreading and repair
- 3’ to 5’ exonuclease occurs during synthesis
- Strand-directed mismatch repair occurs just after synthesis
3’ to 5’ exonuclease activity
Removes misincorporated nucleotide; DNA polymerase moves backwards one spot and clips off one nucleotide (only happens on end of strand)
DNA polymerase two domains
- Polymerizing
- Editing
Strand-directed mismatch repair
- MutS recognizes and locks on to mismatch
- MutS recruits MutL and scans DNA
- strand with error is removed
- gap filled in by polymerase sigma
Pyrimidine dimer
2 covalent bonds with a strand of DNA (TT/CC/CT)
- DNA polymerase can’t move through, either skips area or makes a mistake
2 mechanisms of DNA repair
- Base excision repair
- one nucleotide is fixed - Nucleotide excision repair
- bigger section cut out
- needs helicase
2 types of repair of double-stranded breaks
Nonhomologous end joining
- break repaired with some loss of nucleotides
Homologous recombination
- break repaired with no loss of nucleotides
Molecular definition of a gene, two types of genes
The entire nucleic acid sequence necessary for the synthesis of a protein or RNA
RNA encodes a protein or RNA that functions as RNA
RNA transcript
- only one template strand, ssDNA
- RNA polymerase doesn’t need a primer
Transcription steps in prokaryotes
- Sigma factor binds to RNAP and finds promoter (several failed attempts)
- Unwinding
- Elongation
- Termination
Promoter sequence is __________ from the +1 site
Upstream (more negative number)
RNA Terminator Sequences
GC rich area which forms hairpin followed by AT rich area
- disrupt H-bonding of new mRNA to help dissociate RNA transcript from polymerase
Transcription factors (eukaryotes only)
Proteins that help position eukaryotic RNAPs at the promoter (similar to sigma submit of bacterial RNAPs)
How is RNA polymerase ll activated?
Phosphorylation inducing conformational change to shed general transcription factors
Eukaryotic mRNA processing
- addition of 5’ cap to protect RNA from exonucleases
- removal of introns by rRNA splicing introns into lariats, requires snRNPs
- processing and polyadenylation of 3’ tail
Alternative RNA splicing
Increases the number of gene products
Where is mRNA translated
Cytoplasm, site of protein synthesis
Redundancy
Multiple codons for most amino acids (third nucleotide most flexible)
Types of mutation
Silent
- same amino acid due to redundancy
Missense
- different amino acid
Nonsense
- premature stop codon
Two steps in ensuring fidelity, error correction
- Aminoacyl tRNA synthetases
- Base pairing
Hydrolysis editing
tRNA structure
Aminoacyl site
Peptidyl site
Exit site
Clover leaf shape
Shine-Dalgarno sequences and polycistronic mRNA occurs in…
Prokaryotes only
Translation release factor
Protein, not RNA
