Genetics Exam 3

rRNA

forms ribosome

tRNA

translational RNA

snRNA

RNA splicing

lncRNA

gene silencing

miRNA

post-transcriptional gene silencing

siRNA

breaking down RNA from viruses

+1 notation in transcription

start of transcription

factor independent termination

poly-U stretch at 3` UTR of growing RNA slows transcription and weak hydrogen bond causes RNA polymerase to fall off; GC rich RNA hairpin structure protects rest of RNA

what is a rho and what is rho dependent termination

a helicase protein; C-rich binding site in 3` UTR for Rho protein whose recruitment breaks apart DNA:RNA hybrid, releasing RNA

Why is transcription in eukaryotes more complex?

1. eukaryotes have more genes that are spaced far apart

2. eukaryotes have 3 RNA polymerases which transcribe different sets of genes

3. DNA in eukaryotes is in the form of chromatin, and heterochromatin can’t be transcribed

4. eukaryote RNA has more processing steps

5. transcription in eukaryotes takes place in the nucleus

RNA modification: capping and it purpose

m7G cap is added to 5` end of growing RNA to protect RNA from decay and promotes translation of RNA into proteins

polyadenylation signal

highly conserved sequence at end; A is added to 3` end of mRNA by Poly(A) polymerase, creating poly A tail

what does poly A tail do and what happens once it completes its purpose?

protect mRNA from decay, promotes nuclear export, and promotes protein synthesis; poly-A tail is then recognized and cut off

mRNA splicing and what is this process mediated by?

introns are cut out as non-coding regions; spliceosomes

alternative splicing

the same sequences are not always exons or not always introns, resulting in a different protein

ribosome

mediates mRNA translation into protein

Amino acid structure

1. alpha carbon

2. carboxyl group

3. R group

4. amino group

5. 1 hydrogen

which parts of an amino acid does a peptide form between?

carboxyl and amino

primary structure

order of amino acids

secondary structure

alpha helix, beta sheet

tertiary structure

how R groups interact with each other to form 3D structure

quaternary structure

multiple polypeptides come together to form a complex

RNA → protein steps

1. linear sequence of nucleotides in a gene determines linear sequence of amino acids

2. codon of 3 nucleotides specifies an amino acid

3. genetic code is degenerate

4. each nucleotide is part of only one codon

5. code from fixed starting point and continues until end of reading frame

degenerate

more than one codon can specify the same amino acid

why would an overlapping code be bad? (two reasons)

1. change reading frame

2. mutations would be propagated in multiple amino acids

which bases are more likely to mutate into stop codons (general)?

codons that are one nucleotide off from a stop sequence

How does amino acid attach to tRNA

1. amino-acyl synthetase uses ATP to transfer AA to ATP and tRNA

2. amino acid attaches to tRNA at the synthetase

3. ATP —> AMP

4. amino acid attaches to OH on tRNA 3` end

Initiation steps

1. 30s subunit of ribosome starts process by binding to shine-dalgarno sequence or kozak sequence

2. sequence binds to 16s RNA to align small subunit

3. initiation factors IF1 and IF3 block A and E sites to prevent further binding of tRNA

4. GTP hydrolysis releases IF1,2,3 and allows large subunit to bind, completing assembly of ribosome

what is the difference between the shine-dalgarno sequence and kozak sequence?

shine-dalgarno is in prokaryotes and kozac is in eukaryotes

Elongation steps

1. EF-Tu brings charged tRNAs to A site to join polypeptide

2. EF-G binds in A site and promotes translocation of tRNAs from the P and A sites to E and P sites

What is thought to be the purpose of eEF1 and eEF2 in translation?

thought to aid in proofreading to ensure correct codon-anticodon interactions

Termination steps

1. RF 1 and 3 bind to stop codons and release polypeptide chain from tRNA in P site

   1. ribosome release factors recycle ribosome to begin another round of translation

kinases

enzymes that transfer a phosphate from ATP to an amino acid

phosphatases

enzymes that remove phosphate groups

signaling cascade

phosphorylated receptor recruits additional signaling molecules that activate downstream of kinases; can alter gene expression patterns

signal sequence

first ~20 AA may contain signal peptide that stops translation before allowing protein to be threaded into ER

nuclear localization sequence

short hydrophobic sequences that allow protein to be ferried into nucleus

ubiquitinylation

addition of small protein ubiquitin which targets protein to proteasome for destruction

cDNA and its purpose

complementary DNA that doesn’t contain introns; used for euk gene isolation

in vitro

outside of living organism

in vivo

inside of living organism

steps of molecular cloning

1. use a vector such as plasmid to insert DNA of target gene into E coli

2. use restriction enzymes that cut vector DNA at specific sequences to produce fragments with staggered or blunt ends to create an opening to insert target genes

3. digest vector and target gene (“insert”) with same restriction enzymes to create compatible ends that can anneal

4. incubate vector and insert with DNA ligase to ligate ends

what is the result of molecular cloning?

recombinants that include target gene where restriction site used to be

what if a vector is cut by two restriction enzymes?

ends are different, so the insert would only fit one way

polymerase chain reaction (PCR) steps

1. denature the DNA to separate the two complementary strands, which allows both of them to be used as a template

2. DNA primers anneal target sequence

3. Taq extends DNA strand by adding dNTPs (A,T,C,G)

what is the purpose of PCR?

amplify and make many copies of target DNA

qPCR

measures amount of a specific DNA product in a sample after each cycle using fluorescence

in qPCR, what does it mean when there’s more fluorescence?

there’s more DNA present in the sample

PCR output

how many PCR cycles it takes to reach a fuorescence threshold

C_t value and its meaning

cycle threshold; lower value=more DNA=more fluorescence

Gel electrophoresis

agarose gel separates DNA/RNA by size, with small fragments running through pores faster than larger fragments toward the positive cathode

what does PCR tell you?

presence/absence of gene and size of gene

what does PCR not tell you?

location of gene expression in the cell or expression levels

what does qPCR not tell you?

size of gene, location of gene expression, protein level/location in cell

what does western blot tell you?

size of protein, relative levels, modifications that affect size

what does western blot not tell you?

location of protein in cell, gene expression level

what does in situ hybridization tell you?

location of RNA/DNA in a cell/tissue, relative levels

what does in situ hybridization not tell you?

protein level

what does immunofluorescence not tell you?

gene expression level

what does immunofluorescence tell you?

location of proteins in a cell/tissue, relative protein levels

what do southern blots detect?

DNA

what do northern blots detect?

RNA

what gel does PCR use and why?

agarose gel; it’s looser and thus better suited for larger fragments

what gel do blots use and why?

polacrylamide gel; it’s tighter so it gives better accuracy for the small molecules

western blot antibodies

primary: detects protein

secondary: detects primary antibody and fluoresces

why is cDNA easier to work with?

it is shorter due to its lack of introns

Sanger dideoxy DNA sequencing

1. ddNTPs are incorporated into growing DNA chain to halt extension

2. 4 reactions are needed, each with a single ddNTP

3. separate chains by size on a gel and length will tell you where chain got terminated

shotgun sequencing steps

1. break genomic DNA into smaller pieces that are cloned, then sequenced

2. sequence each fragment and find overlaps

3. align overlaps and combine them into contigs that can be overlapped into a genome

Next generation sequencing

1. cut DNA into small fragments

2. adapter sequences are attached to ends of fragments and are unique to each end

3. fragments are bound to flow cell with oligonucleotides complementary to adapter sequences; adapters bind to their complements, forming a bridge

4. each bridge is amplified by PCR

5. computer detects which base is added during synthesis (sequencing by synthesis)

why are short reads bad for repetitive sequences?

they look the same (ambiguous) so computer can’t detect them

paired-end sequencing

a way to join two contigs by scaffolding them next to each other; also helpful for repetitive sections

ChIP-Seq Steps

1. cross-link proteins to DNA

2. antibody used to retrieve protein and any associated DNA

3. crosslinks are broken and associated DNA is sequenced

4. sequencing comes back as overlapping DNA sequences (reads) that are then mapped to the genome to see what sequences were present in that sample

what does the number of reads indicate in ChIP-Seq?

directly proportional to strength of interactions

Mass spectrometry use

used to determine composition of sample and all proteins present

mass spectrometry steps

1. run mixture of proteins on a gel

2. digest into peptides

3. ionized and shown relative abundance`

what does mass spectrometry not tell you?

location, function, DNA/RNA sequence, gene expression levels

electroportation

electrical pulse creates small holes in lipid bilayer that allows DNA to pass

forward genetics

to pinpoint which gene causes a particular phenotype

reverse genetics

to pinpoint which phenotype is caused by a particular gene

chimera

an organism with DNA from two different sources

sgRNA

20 nucleotide RNA designed by researcher, close to region of interest and near a PAM and Cas9 endonuclease

what is a PAM?

a nucleotide followed by two guanines

what is the purpose of Cas9?

it will cause a double stranded break that’s improperly repaired and creates nonfunctional gene

activators

positively regulates transcription

repressors

negatively regulates transcription by binding to operator and preventing polymerase from moving forward

allosteric binding

effector binds to site on protein that isn’t activation site, causing a shape change

lac operator: cis or trans and what does it mean?

cis; it cannot activate transcription on a different chromosome

how does lac operon work?

• in absence of lactose, lac operon is repressed

• in presence of lactose, lactose binds allosterically to repressor, changing its shape and causing it to detach from the operon

lac repressor: cis or trans and what does it mean?

trans; it can repress the lac operon of either of the homologs

what happens to the lac operon when ATP levels are low?

cAMP and CAP bind together on the lac operon to promote transcription, hoping lactose in the environment can be taken up

cAMP and lactose levels low, no CAP, lac repressor bound:

no transcription

cAMP levels low, lactose levels high, no CAP, and unbound lac repressor:

low gene expression

cAMP levels high, lactose levels high, CAP present, no repressor:

high gene expression

attenuate

premature stopping of translation

trp operon

• has 14 amino acid sequence, including 2 trp codons and STOP

• acts as a sensor to see how much trp is present

what happens when trp levels are high?

• there are more trp tRNAs for quick transcription

• favors terminator loop (3+4)

  • too much so stop

what happens when trp levels are low?

• fewer trp tRNAs for slower transcription

• favors anti-terminator loop (2+3)

  • too little so go

transcription factors and where do they bind?

DNA-binding proteins that activate/repress gene expression of specific types of genes; bind to enhancers

general transcription factors

DNA-binding protein that binds to promoter to activate gene expression of most genes

mediator

used to connect enhancer to promoter when they’re far apart