gene expression

northern blot

transfer gel to membrane, DNA probe

RTPCR

RNA—>cDNA, then PCR

qRTPCR

same as rtpcr quantitative pcr machine measures amt of transcript.

RNA sequencing

RNA—>cDNA done through reverse transcriptase

Probes

fragment of single strand dna that finds and binds with matches. this is hybridization.

SYBR green

qpcr fluorescent dye

what is needed for qRTPCR?

loading control and experimental control

Next gen sequencing

for large amt of small dna fragments at once.

capping molecule

7-methylguanosine

CTD

c-terminal domain, RNA pol 2. landing platform that recruits proteins. capping splicing, polyadenylation. phos determines recruited protein.

spliceosome

made of snRNAs and proteins. uses u1-u6

splicing mechanism

• branch 2’ OH attacks 5’ splice site

• severed 5’ exon activated to attack 3’ splice site

• Intron released as lariat

cis acting

located on same molecule

trans acting

mobile, target genes

cis/trans experiment

add 2nd copy of gene on plasmid

• works=cis mutation

• fails=trans mutation

lac operon

NEEDS l and o to regulate, z,y, and a metabolize.

lactose effects

lactose: repressor falls off, transcription on

no lactose: repressor binds to allolactose, transcription off

allolactose

allows transcription, increases it.

glucose

glu: cAMP goes down, WEAK transcription

no glu: cAMP goes up, binds CRP, transcription goes up

no lac z

poor induction

structure motifs

• helix-turn-helix= grab

• zinc finger= finger support

• leucine zipper= zips

HAT

add acetyl, which increases transcription

HDAC

removes acetyl, decreases transcription

SWI/SWF

nucleosome moving/remodeling

Genes On

1. transcription factor binds

2. HATS are recruited to acetylate histone tails

3. nucleosome remodeling to expose promoter

4. chromatin opens

5. mediator and regulators bend DNA to loops

6. transcription machinery recruited

7. transcription

8. HDAC de-acetylates

mello and fire

can rna silence genes?

• used mex-3 in worms

• added antisense (single strand rna) and dsRNA (both strands together)

What did they measure?

• stain-mex-3 expression, results:

No RNA Antisense RNA dsRNA

normal mex-3 weak reduction strong silencing

What does this mean?

dsRNA triggers RNA interference!

Explains miRNA

miRNA

small RNAs that silence genes. natural.

• bind mRNA and prevent protein production

how are miRNA made?

1. pri-miRNA: Long mRNA transcribed, folds into hairpin

2. Drosha: cuts hairpin into pre-miRNA in nucleus

3. Export to cytoplasm

4. Dicer cuts it into short dsDNA

5. RNA helicase separates strands

6. RISC: complex that mature miRNA is loaded into

how does miRNA silence genes?

perfect match: mRNA degraded

partial match: translation blocked

Drosha

cuts pri-miRNA

Dicer

cuts dsRNA smaller

RISC

silences target mRNA. (miRNA inside)

siRNA

binds to and degrades mRNA, near perfect match, artificially added.

created as single OR double strand

perfect/partial

perfect: dsRNA. mRNA is rapidly degraded

partial: RISC, physically blocks translation

how does miRNA recognize?

binds seqs in 3’ UTR of mRNAs, can target many genes. a gene can have many miRNA seqs.

shRNA

artifical, placed into plasmid, forms hairpin to be processed into miRNA machinery. starts as DNA sequence