BIMM M2

What is the overall flow of gene expression?

DNA → transcription → RNA processing → mature mRNA → translation → protein

What levels can regulate gene expression?

Chromatin accessibility, transcription, RNA processing, translation

What enzyme synthesizes RNA during transcription?

RNA polymerase

In what direction is RNA synthesized?

5’→3’

In what direction is the template strand read?

3’→5’

How does the coding strand compare to RNA?

Same sequence except T instead of U

Does RNA polymerase require a primer?

No

What nucleotides are used in transcription?

rNTPs

What are the four rNTPs?

ATP, UTP, GTP, CTP

What does mRNA encode?

Proteins

What is the role of rRNA?

Forms part of the ribosome

What is the role of tRNA?

Translation adapter molecule

Are most RNAs protein coding?

No

Where does RNA polymerase bind?

Promoter

What helps RNA polymerase recognize promoters in prokaryotes?

Sigma factor

Are sigma factors found in eukaryotes?

No

What happens to sigma factor after initiation?

It dissociates

What happens in the lac operon when lactose is low?

LacI binds operator and transcription is OFF

What molecule inactivates LacI?

Allolactose

What converts lactose into allolactose?

Beta-galactosidase

What happens when glucose levels are low?

cAMP levels increase

What does cAMP bind?

CAP

What does CAP do?

Activates transcription

When is lac operon transcription strongest?

High lactose and low glucose

What forms during intrinsic termination?

GC-rich RNA hairpin

What follows the hairpin in intrinsic termination?

U-rich region

What happens during rho-dependent termination?

Rho binds RNA and catches RNA polymerase

What are histones?

Proteins DNA wraps around

What is a nucleosome?

DNA wrapped around histones

What histones form the octamer?

2x H2A, 2x H2B, 2x H3, 2x H4

What does H1 bind?

Linker DNA

What is open chromatin?

Accessible and transcriptionally active chromatin

What is closed chromatin?

Condensed and inactive chromatin

Are eukaryotic genes ON or OFF by default?

OFF

Are prokaryotic genes ON or OFF by default?

ON

Why are eukaryotic genes usually OFF by default?

DNA is packaged into chromatin

What charge does DNA have?

Negative

What charge do histones have?

Positive

What type of interaction occurs between DNA and histones?

Electrostatic interactions

Do histones bind DNA sequence specifically?

No

What part of DNA do histones interact with?

Phosphodiester backbone

What does histone acetylation do?

Opens chromatin

What enzyme acetylates histones?

Histone acetyltransferase (HAT)

What does acetylation do to histone-DNA interactions?

Weakens them

Can methylation activate or repress chromatin?

Yes

What modification is associated with active transcription?

H3K4me3

What modification is associated with heterochromatin?

H3K9me3

What are pioneer factors?

Transcription factors that bind closed chromatin and open it

What does DNase-seq measure?

Open chromatin

What does DNase cut?

Accessible DNA

What do DNase-seq peaks indicate?

Open/accessibile chromatin

What does ChIP-seq measure?

Protein or histone modification binding locations

What does RNA-seq measure?

Gene expression

What do more RNA-seq reads indicate?

More gene expression

What does ATAC-seq measure?

Open chromatin

What does ATAC-seq use?

Transposase

What is the difference between DNase-seq and ATAC-seq?

DNase-seq uses DNase, ATAC-seq uses transposase

What is the difference between DNase-seq and ChIP-seq?

DNase-seq measures accessibility, ChIP-seq measures protein/histone binding

What polymerase synthesizes mRNA in eukaryotes?

RNA polymerase II

What polymerase synthesizes rRNA?

RNA polymerase I

What polymerase synthesizes tRNA?

RNA polymerase III

What binds the TATA box?

TBP

What complex contains TBP?

TFIID

What does TFIIH do?

Unwinds DNA and phosphorylates RNA Pol II CTD

What does NELF do?

Pauses RNA polymerase II

What releases NELF?

Phosphorylation

What initiates eukaryotic transcription termination?

Recognition of the poly(A) signal

What happens after poly(A) signal recognition?

RNA cleavage and poly(A) tail addition

What do enhancers do?

Increase transcription

What do activators do?

Increase transcription

What do repressors do?

Decrease transcription

How can enhancers act from far away?

DNA looping

What determines whether enhancer mutations affect transcription?

Changes in transcription factor binding

What is added to the 5’ end of eukaryotic mRNA?

7-methylguanosine cap

Functions of the 5’ cap?

Protection, export, translation initiation

What is removed during splicing?

Introns

What is joined during splicing?

Exons

What initiates spliceosome-mediated splicing?

2’-OH of intron adenosine

What attacks the 5’ phosphate of exon 2 during spliceosome splicing?

3’-OH of exon 1

What initiates type I self-splicing?

External guanosine 3’-OH

What initiates type II self-splicing?

Intron adenosine 2’-OH

What do all splicing mechanisms use?

Nucleophilic attack reactions

What is alternative splicing?

One gene producing multiple proteins

What enzyme adds the poly(A) tail?

Poly(A) polymerase

Functions of the poly(A) tail?

Stability, export, translation efficiency

What is the difference between the poly(A) signal and poly(A) tail?

Signal triggers cleavage, tail is added afterward

What does mature mRNA contain?

5’ cap, exons, UTRs, coding region, poly(A) tail

What is translation?

mRNA → protein

In what direction does protein grow?

N-terminus → C-terminus

What is the role of tRNA?

Matches anticodon to codon and carries amino acid

What is a codon?

3 nucleotides in mRNA

What is an anticodon?

3 nucleotides in tRNA

Where does wobble occur?

5’ end of anticodon

What can G pair with in wobble?

C or U

What can U pair with in wobble?

A or G

What can inosine pair with?

A, U, or C

Why is wobble important?

Allows fewer tRNAs

What does redundant genetic code mean?

Multiple codons code for same amino acid

What does universal genetic code mean?

Same codons used across organisms

What is the start codon?

AUG