Cellular and molecular bio quiz 2

def for DNA replication

copying a double stranded (ds) DNA double helix.

what direction is DNA synthesized?

5’ to 3’

replication origin, rep fork, DNA POL 3, DNA helicase, Single-Strand DNA binding protein, DNA topoisomerase, sliding clamp, clamp loader, leading strand, lagging strand, primase, Okazaki fragments, DNA POL 1 DNA Ligase, telomerase

rep origin: where DNA replication begins

rep fork: created when DNA helicases unwind the DNA stands

DNA POL3: catalyzes the addition of nucleotides to the 3’ end of a growing strand, Uses a template and makes a complimentary strand proof reads work.

Single-Strand DNA binding protein: Binds to single stranded DNA exposed by DNA helicase

DNA topoisomerase: helps relive tension in one strand of the DNA double helix built up by unwinding the DNA by breaking the DNA then resealing the breaks. in front of the rep fork

Sliding Clamp: Keeps DNA Pol attached to the template so it does not fall off the DNA

Clamp loader: Uses the energy of ATP hydrolysis to lock the sliding clamp onto the DNA

leading strand: elongated continuously toward replication fork

lagging strand: elongated discontinuously, away from replication fork

primase: Synthesizes RNA primers along lagging-strand template

Okazaki fragments: Fragments of DNA on the lagging strand

DNA POL 1: replaces RNA primer with DNA 5’ to 3l

DNA ligase: Uses ATP hydrolysis to seal nick between Okazaki fragments

Telomerase: replicates the end of eukaryotic chromosomes

Endogenous sources, exogenous sources, nucleotide excision repair, mismatch repair

endogenous sources: originating w/in cell – e.g., replication, chemical reactions

exogenous sources: originating in the environment – e.g., ionizing radiation, UV light, chemicals

nucleotide excision repair: fixes damaged nucleotides. Segment of damaged DNA excised, repair DNA pol fills in missing nucleotide using other stand as template, DNA ligase seals nick

\mismatch repair: fixes mismatched nucleotides; proofreading errors

Error rates of DNA replication (no proofreading), DNA replication (with proofreading, no mismatch repair), DNA replication (both)

1. one mistake per 10^5 nucleotides copied

2. one mistake per 10^7 nucleotides copied

3. one mistake per 10^9 nucleotides copied

non-homologous end joining, homolgous recombination

non-homologous end joining: DNA ligase rejoins DNA in double strand break, some nucleotides lost but faster

homologous recombination: DNA pol comes in and codes the missing sequences, Ligase joins it together, slower.

gene, structural gene, transcription, translation

gene: sequence of DNA that codes for a functional product

structural DNA: codes for a protein

transcription: DNA sequence of the coding region of a gene copied into messenger RNA (mRNA)

Translation: sequence of mRNA specifies a sequence of amino acids in a polypeptide (protein)

Summary of Important Differences Between DNA & RNA

messenger RNA, Transfer RNA, Ribosomal RNA, small nuclear RNA, microRNA

mRNA: transcript used to synthesize proteins

tRNA: adapter between mRNA codons and polypeptide amino acids

rRNA: RNA component of ribosomes

snRNA: RNA component of spliceosomes

miRNA: involved with the regulation of gene expression

Transcription, RNA POL

Transcription: copying information in DNA into a temporary RNA molecule

RNA POL: enzyme of transcription, synthesizes complimentary RNA from DNA template strand, elongates 5’ to 3’, no proofreading, no primer, opens <20 bp to access template, no helicase allows for amplification of the DNA information

Prokaryote transcription:

Initiation: – promoter: just upstream of gene coding

region • specific sequences at -10, -30 – sigma (浀) factor: RNA pol subunit, binds promoter sequences

• Elongation: – RNA pol adds complementary RNA nucleotides from transcription start site: +1 – sigma factor released

• Termination: – terminator: downstream, specific sequences signal RNA pol release

– included as part of transcription

Transcription in eukaryotes

RNA pol II: for protein coding genes • multiple general transcription factors needed with RNA pol II= transcription initiation complex – assemble at promoter – multiple binding sequences, including TATA box – TFIID (with TATA binding protein, TBP) finds promoter – other TFs & RNA pol II assemble – RNA pol II tail phosphorylated: released from complex to transcribe Elongation factors allow RNA polymerase II to move through DNA packaged in

nucleosomes.

How is precursor mRNA packaged and made ready? defs for RNA capping, Polyadenylation, RNA splicing, exons, introns, spliceosome

RNA capping: adds 7- methylguanosine + 3 phosphates to 5’ end, “5’ m7G cap” • added as mRNA is produced

Polyadenylation: 100s of A’s added to 3’ end, “poly-A tail”

RNA splicing: splicing exons, removing introns

exons: expressed sequences

introns: intervening sequences, recognized by specific sequences at their ends

spliceosome: large protein/RNA machine that assembles to remove introns, splice exons

alternative splicing

Alternative splicing: allows for more than one protein to be produced from the same gene by choosing which exons to keep

RNA processing in euks and proks

Euk RNA processing: provides binding sites for proteins mark mRNAs for export through nuclear pores to cytosol – mark mRNAs to not be degraded – exported for translation in cytosol • in euk., kept for 10-30 min • degraded when done

Bacteria RNA processing: mRNAs translated immediately – mRNAs kept for 3 min

how many ways can an mRNA molecule be translated?

three possible reading frames

mRNA, codons, genetic code, ribosome, tRNA, anticodons, polypeptide

mRNA: messenger RNA

Codons: mRNA nucleotide triplets specify amino acids

Genetic code: correspondence among codons and amino acids

Ribosome: site of translation

tRNA: transfer RNA

Anticodons: complement codons – bound to amino acid

polypeptide: chain of amino acids

ribosome, large subunit, small subunit, ribozymes

ribosome: organelle of protein synthesis – composed of ribosomal proteins and ribosomal RNA (rRNA)

large subunit: 49 proteins and 3 rRNAs (28S, 5.8S, 5S) – 3 tRNA binding sites (E, P, A); catalyzes peptide bond

small subunit: 33 proteins, 18S rRNA – binding site for mRNA; matches codons of mRNA & anticodons of tRNA

ribozymes= RNA enzymes – 2/3 rRNA by mass, gives structure

five steps of elongation of polypeptide during translation

1. charged tRNA with amino acid in the A site, tRNA with growing polypeptide in the P site - anticodons complement codons

2. C-terminus of polypeptide uncoupled from tRNA in P site, transferred to amino acid in the A site - peptide bond catalyzed by large subunit

3. large subunit shifts one codon in the mRNA 3’ direction - spent tRNA in E site - tRNA with polypeptide in the P site

4. small subunit shifts one codon in the mRNA 3’ direction - tRNA in E site ejected

5. next charged tRNA corresponding to next mRNA codon enters A site

Initiation and termination of translation

Initiation: – initiator tRNA = initiator methionine: has anticodon complementary to start codon (AUG) – Translation Initiator factors: proteins match initiator, P site of small ribosomal subunit, and 5’ cap of mRNA – searches mRNA toward 3’ for start codon – large subunit joins assembly – translation proceeds

Termination: stop codons (UAA, UAG, UGA) have no tRNA – release factor: protein hydrolyzes polypeptide from last tRNA – whole thing disassembles

Genetic code, start codon, stop codons, wobble

Genetic Code: the key to translating between mRNA codons and amino acids, has redundancy

Start codon: AUG – also methionine

•Stop codons: UAA, UAG, UGA

wobble: some tRNAs pair with multiple codons

Gene expression can be regulated at multiple steps in the pathway

DNA access: heterochromatin, epigenetic control

transcription: transcription factors

post-transcription: 5’ cap, poly-A tail, exon splicing, transport to cytoplasm

translation: degradation of mRNA, inhibition by miRNA, other translational control

post-translation:, modification e.g., phosphorylation, cleavage, combination with other polypeptides, transport to cellular target

defs for housekeeping genes, transcription regulators, chromatin-remolding complexes

HG: genes expressed in all cells

RT: proteins that affect formation of the transcription initiation complex

Chromatin-remodeling complexes: determine DNA accessibility

Gene expression can be regulated at various steps from DNA to RNA to

Protein

defs for operon, operator, repressor, trp repressor

operon: coordinated cluster of genes, common in bacteria and a single promoter

operator: Regulatory DNA sequence in promoter

repressor: binds to operator to stop synthesis

trp repressor= allosteric transcription regulator – inhibited by tryptophan • Trp high, active repressor binds operator: blocks RNA pol • Trp low, inactive repressor; RNA pol transcribes

lac operon steps include lac repressor and CAP activator

def for enhancers, activators, mediators, transcriptional regulators, looped domains, combinational control, epigenetic inheritance

enhancers: Regulatory DNA sequence in eukaryotes, 100s of bps away from promoter, bound by activators and also repressors

activators: protein that binds to an enhancer transcription regulators, AKA specific transcription factors

mediators: complex of proteins between activators and the transcription initiation complex (general transcription factors and RNA polymerase)

transcriptional regulators: attract proteins that remodel chromatin – tightly-bundled chromatin inhibits transcription – histone modifying enzymes: e.g., attaches acetyl groups to histones • provide binding sites for general transcription factors, etc. – chromatin-remodeling complex: make regulatory DNA sequences more accessible

looped domains: regulate the associations between enhancers and their genes

CC: multiple transcription regulators operate to control expression – common in eukaryotes – multiple transcription regulators per gene – each transcription regulator interacts with multiple genes= coordination

epigenetic inheritance: transmission of traits not caused by changes to the genetic sequence. i.e. “outside of the genome

do somatic cells in a multicellular organism contain the same DNA?

yes

all sources of mutations in an organism

within a gene: change DNA, change RNA, change protein

– in regulatory DNA: change DNA, change where/when gene expressed

– gene duplication: copied gene can evolve new function

– exon shuffling: swapping protein domains, new proteins with new functions

– transposition: mobile genetic elements contribute mutations

– horizontal transfer: DNA passed from one cell to another

– genome duplication: diploid (2n), tetraploid (4n), octoploid (8n), etc.

mutations in asexual vs sexual reproduction

asexual: all mutations passes

sexual: only germ line (gamete cells) mutations pass

point mutation, neutral mutations, silent mutations, missense mutations, deleterious mutations, nonsense mutations

PM: replace one nucleotide with another

NM: no effect on fitness

SM: change codon but not amino acid

MM: change aa but not protein function

deleterious: decreases fitness

NM: changes codon to stop codon

gene duplication can create new genes, crossing over, unequal crossing over

misalignment during homologous recombination leads to gene duplications & deletions

crossing over: swaps segments between homologous chromosomes

unequal crossing over: one homologous chromosome gets part of the other

exon shuffling, mobile genetic elements/transposable elements/transposons, DNA only transposons, nonrepetitive DNA, repetitive DNA

EX: combining exons from different genes

transposons: segments of DNA that can move in the genome

DNA only: mobile elements without an RNA intermediate

NR DNA: 28% of the human genome regulatory DNA, RNAs, pseudogenes

R DNA:50% of the human genome, occurs in multiple copies in the genome

how do we find recognizable similarity between bacteria, archaea and eucarya genes?

small ribosomal rRNA gene

how much of the human genome codes for proteins?

2%

Two major families of transposable sequences in the human genome

1: DNA only transposons like bacteria

2: retrotransposons= mobile elements with an RNA intermediate – only in eukaryotes (most common type in humans) – transcribed into RNA by RNA pol – new DNA made from RNA template by reverse transcriptase – integrated in new location: “copy & paste

viruses and what they are made of (genome, capsid, viral envelope)

virus: transferred from cell to cell, use host cellular machinery to express genes and replica

genome: DNA or RNA ss or ds

capsid: protein coat

viral envelope: derives from host plasma membrane

retrovirus

A virus with RNA genome and DNA stage, like HIV

ss RNA genome – reverse transcriptase uses RNA to make ds DNA – ds DNA inserted into host chromosome – can be latent for years: integrated into genome and inactive – completes life cycle when transcribed

DNA sequence, DNA amplification, gel electrophoresis, DNA ladder, PCR

DNA sequence: the list of nucleotides of a segment of DNA

amplification: lost of copies of DNA

gel electrophoresis: uses porous matrix (e.g., agarose) and electricity to separate DNA by size – short molecules move faster than long ones

DNA ladder: standard fragments of known length

PCR: used to amplify specific DNA sequences.

how to isolate DNA molecules

isolate target DNA from unwanted DNA with restriction enzymes and gel electrophoresis

– generate a recombinant DNA molecule

– determine the order of nucleotides in each DNA helix via sequencing

whats needed for PCR

Dideoxy sequencing

shotgun sequencing, RNA-seq, meta-genome, reporter gene, transgenic organism, gene knockout

shotgun sequencing: includes lots of different methods – divide genome into lots of small, random fragments – amplify and sequence those fragments – assemble contiguous fragments based on overlapping sequences

RNA-seq: use cDNA library or direct RNA sequencing (dRNA-seq) to find expressed genes

meta-genome: collective sequences of one gene from an environmental sample

– e.g., identify all the bacteria in a sample by sequencing all the 16S (small ribosomal subunit) DNA

reporter gene: insert gene for easily visualized product after gene’s promoter, Allow Specific Proteins to Be Tracked in Living Cells

transgenic organism: genome altered by stably inserting gene

– e.g., replace gene with mutant version in embryonic stem cells, get cells into germ line • difficult and time consuming

gene knockout: turn off or mutate gene of interest

CRISPR, cas, cas9

CRISPR:: bacteria anti-viral system – clustered regularly interspaced short palindromic repeats – between repeats: copies of viral (phage) genomes – transcribed: palindromic seq. makes stem-loops in guide RNA

Cas (CRISPR associated) endonuclease: cut complementary viral DNA based on guide RNA

cas9: engineered endonuclease that uses guide RNA to cut complementary ds DNA – cut ends ligated with random nt between them: “knockout” gene – OR, if template DNA present, repair enzymes can insert transgene

plasma membrane, internal membrane, three components of cell membrane (lipid bilayer, membrane proteins, carbohydrates), hydrophilic and hydrophobic molecules

PM: outer cell membrane surrounding the entire cell

IM: enclose compartments/organelles within the cell

lipid bilayer: hydrophobic permeability barrier, provides structure

membrane proteins: embedded and adjacent proteins

carbohydrates: attached to membrane lipids and proteins

hydrophilic molecules: charged or polar, able to hydrogen bond with water – dissolve in water – e.g., salts, sugars, nucleic acid

hydrophobic: nonpolar molecules disrupt hydrogen bonds with water – hydrophobic force: minimizes interactions between hydrophobic and hydrophilic molecules – e.g., fats, oils, waxes, steroids

amphipathic, phospholipids, cholesterol, glycolipids

amphipathic: molecules that are hydrophobic and hydrophilic with a hydrophilic polar head and a nonpolar hydrophobic tail

phospholipids: most abundant membrane lipids – charged phosphate “head” – two hydrocarbon “tails”

cholesterol: n animal cells, esp. plasma membrane (20% lipids by mass)

glycolipids: lipids with attached sugars, carbohydrates

what forms spontaneously in an aqueous solution?

lipid bilayer

lipid bilayer, fluid

LB: 2 monolayers of amphipathic lipid molecules – hydrophilic “heads” contact aqueous solution – hydrophobic “tails” contact each other – energetically favorable arrangement - forms a sphere, no exposed edges

fluid: individual phospholipids capable of motion – lateral diffusion, rotation, flexion – but NOT flip-flopping between monolayers

what do phospholipid bilayers do?

spontaneously close in on themselves to form sealed compartments.

fluidity of the bilayer and temperature

membrane assembly steps and defs for lumen, scramblase, flippases

lumen: space inside an organelle

scramblase: enzyme randomly flip-flops phospholipids to even-out

flippases: transporter proteins flip- flop specific phospholipid types to the cytosolic monolayer

True or False? Certain Phospholipids Are Confined to One Side of the

Membrane.

True, Membranes retain their orientation during transfer between cell compartments= the endomembrane system. – Allows for integral membrane proteins and protein secretion.

membrane proteins, {transporters, channels}, anchors, receptors, enzymes, integral membrane proteins, transmembrane proteins, peripheral membrane protiens

membrane proteins: associated with membranes, 50% of mass, 50:1 lipids:proteins

transporters, channels: move small molecules thru membrane

anchors: attach to other macromolecules for stability

receptors: binding external ligand causes change inside cell

enzymes: catalyze reactions

integral membrane proteins: attached to lipid bilayer by hydrophobic forces

transmembrane proteins: have intracellular and extracellular domains

peripheral membrane proteins: noncovalently associate with integral membrane proteins – Extracellular OR cytoplasmic

single-pass transmembrane proteins, multi-pass transmembrane protein

single-pass transmembrane proteins: hydrophobic amino acids on outside of alpha helix; interact with phospholipid tails •

multi-pass transmembrane protein: ≥2 alpha helices – can form a channel – hydrophobic a.a. on outside, hydrophilic a.a. on inside • sometimes beta sheets form into a

beta barrel

cell wall, extracellular matrix, cell cortex

cell wall: rigid carbohydrate structures in bacteria, plants, fungi

extracellular matrix: network of proteins that provides structural support to cells or tissues

cell cortex: internal network of fibers – e.g., spectrin, actin, myosin – peripheral membrane proteins – attach to integral membrane proteins to hold cell shape

glycocalyx, oligosaccharides

Glycocalyx: a layer of carbohydrates surrounding cells

oligosaccharides: (short polymers) attached to glycolipids and glycoproteins – protects plasma membrane – lubricates: hydrophilic, attracts water – involved with cell-cell recognition • e.g., ABO blood type