Lecture 3: Transcription - DNA to RNA

What enzyme does RNA transcription

RNA polymerase

In bacteria, what guides RNA polymerase to the promoter region

sigma factor

In bacteria, once RNA synthesis begins, what happens to sigma factor?

it is released to be used again

How does RNA pol know where to stop?

sequences known as terminators indicate where the gene ends

what polymerase transcribes mRNA specifically

RNA pol II

What does RNA pol II transcribe

mRNA

What factors does RNA pol II work with to initate transcription

1. TFIID

2. TFIIB

3. TBP

4. TFIIA

What is the sequence of events for RNA pol to begin interacting with DNA

1. TFIID recognizes the TATA box (part of the promoter) via the TATA binding protein

2. TFIIB acts like the bacterial sigma factor recognizes other elements of the promoter

3. TFIID and TFIIA/TFIIB create a deformation in the DNA to signal the start of a gene

How does TFIID recognize the TATA box

TATA binding protein (TBP)

What proteins Must interact with the DNA transcription start site before RNA pol II will bind

1. TFIID + TBP

2. TFIIB

3. TFIIA

During transcription, what happens after the initiation factors bind?

transcription initiation complex begins to assemble on the DNA: RNA pol II works with TFIIF and TFIIE to recruit TFIIH

TFIIH

a large multi-protein complex with multiple important roles:

Roles of TFIIH

1. helicase activity to open the DNA strands

2. phosphorylates the C terminal domain of RNA pol II to release from the promoter and allow transcription to begin

What happens to each of the initiator proteins once transcription begins

1. TFIID stays behind to recruit another RNA polymerase

2. other initiator proteins dissociate from RNA pol once transcription begins

3. elongation factors associate with RNA pol II

What is the function of elongation factors during transcription

associate with RNA pol II and keep it associated wiht DNA until reaching a termination site

What is the function of “Mediator” in transcription

it is a large complex that connects RNA pol II with activator proteins on distant enhancer sites

Ahead of RNA polymerase, what loosens DNA from its histomes

histome modifying enzymes

function of mRNAs in cell

code for proteins

function of rRNAs

ribosomal RNA, form the basic structure of the ribosome and catalyze protein synthesis

tRNAs

the adaptors between mRNA and amino acids

telomerase RNA

serves as the template for the telomerase enzyme that extends the ends of chromosomes

Function of RNA pol I and III

transcribe ribosomal rRNA genes

What additional processing steps must eukaryotic mRNA go through as compared to prokaryotes

1. End modifications

2. splicing

3. nuclear transport

How is the 5’ end of mRNA modified?

guanosine cap

how is the 3’ end of mRNA modified

polyadenylated

What does the cell use the 5’ and 3’ modifications for?

to determine if the transcript if complete and a product of self → lack of these modifications can target the mRNA for degradation

Exons

protein coding regions of genes

Introns

non-coding sections of genes that must be removed before translation

Do cells always cut the same introns/exons every time?

No, cells can choose which exons to include depending on the context

splice variants: where do they come from?

come from the same gene but have different exons used to make the final protein

How does a bee larvae turn into a queen

1. The larvae is fed the royal jelly

2. the jelly silences DNMT3 resulting in low DNA methylation

3. 2s intron will not be spliced out of the queen’s mRNA

What residue is involved in attacking the 5’ splice site

adenine

How does the adenine attack of a 5’ splice site work?

at a site close to an intron>exon boundary, an adenine attacks the 5’ splice site, cleaving the sugar phosphate backbone, leabing a hydroxyl group

What happens in mRNA spicing once the free hydroxyl is exposed

the free hydroxyl attacks the 3’ splice site, releasing the intron as a lariat and JOINING the 5’ and 3’ ends together

What assembly mediates the splicing of introns from mRNA

spliceosome

spliceosome

an assembly of RNA and protein molecules that recognizes sites and brings sites close together

What are two examples of what can happen when splicing goes wrong?

1. exon skipping

2. cryptic splice site selection

How do cells choose appropriate splice sites

exon definition: exon size tends to be much more uniform than intron size and this helps the splicing machinery seek out homozygously sized exons rather than intron sequences

How do the capping, splicing and 3’ processing proteins know to associate with the RNA pol II interacting with DNA?

TFIIH phosphorylated RNA pol II and this created placed for these mRNA processing proteins to bind

how are transcriptionally active regions of a chromosome usually arranged?

clustered together

What does the mature mRNA need to have checked before it leaves the nucleus?

that is has a 5’ cap and 3’ poly-A tail → if it passes these checks it will be exported from the nucleus through the nuclear pore complex (if it was not successful, it will be sent to the nuclear RNA exosome)

Where is rRNA transcribed

in the nucleolus

how is rRNA transcribed

as one long transcript that needs to be processed (does not undergo capping or polyadenylation because RNA pol I does not have a phosphorylated tail) → the precursor rRNA is chemically modified and broken apart to form different subunits

What do the locations of rDNA genes in the genome prompt

the formation of a fibrillar center where rRNA is transcribed

can fibrillar centers associate with eachother

yes, they can come together and share other components to assemble complete ribosomes

What holds the nucleolus together????

not a membrane but liquid liquid phase separation!

Steps of RNA seq

1. lyse cells of interest and extract RNA

2. use a technique to isolate the RNA molecules you are interested in

3. RT to create cDNA

4. use primer library to amplify and test sequences to match with known genes

hwo does single cell RNA seq work

similar to RNA seq but individual cells are separated out rather than lumping the entire tissue/population together → helps create a map of cell types in a tissue

on a single-cell RNA seq map, what do the clusters represent

cells with similar transcription profiles that are identified as the same cell type