2. RNA Synthesis- Transcription

IN THIS LECTURE HE SAID HE IS TESTING THE MOST ON REGULATION!!!!! NOT THE ACTUAL STEPS

Central Dogma of Molecular Biology

RNA Polymerase in Prokaryotes:

• Core enzyme/Apoenzyme

• Holoenzyme

• Sigma Factor

• Core enzyme: 2 alpha subunits, beta and beta’ unit and omega unit

• Holoenzyme: core enzyme + sigma factor

• Sigma Factor: Recognizes promoter

RNA Polymerases I, II and III

RNA pol I: codes most rRNA genes

RNA pol II: all protein-coding genes—> mRNA!!! ←TQ

RNA pol III: tRNA, small RNA’s and 5S rRNA genes

mRNA

• what does it correspond to?

• Converted into what?

• In Eukaryotes it is made by which RNA Pol?

• Prokaryotes vs. eukaryotes

• Corresponds to a DNA sequence

• Converted into amino acid sequence during translation

• RNA pol II

• Prokaryotes: Polycistronic, occurs in cytoplasm, not always 5’ cap or 3’ tail, transcription is coupled to translation

• Eukaryotes: Monocistrionic, occurs in nucleus, 5’ cap and 3’ tail, transcription and translation are separate,

tRNA

• Secondary structure

• Functions

• Synthesized by which RNA pol

• Looks like a curved croissant

• Function is to carry amino acids into ribosome to make proteins

  • Anti codon of tRNA reads mRNA codon

• DNA Pol III

rRNA

• What is it?

• What does it do?

• The unit used to measure the amount of sedimentation of ribosomes is

• What DNA pol synthesizes it?

• Ribosomal RNA; make up ribosome organelle

• It makes proteins during translation

• Svedberg unit

• DNA Pol I and III

Prokaryotic Promoter

The most upstream component is:

Next follows the ________ region and then the ________ _____ which ,are recognized by the __ _________

The most upstream component is: the upstream promoter element

Next follows the TTGACA region and then the TATA Box, which are recognized by the sigma factor (of the holoenzyme)

Other Prokaryotic Regulatory Sequences

Operator is located at the end of the promoter

Activator Binding site

Eukaryotic Promoter elements

Short sequence elements that bind transcription factors (sigma factor) and recruit RNA pol

GC box: has increased amount of transcription when there is not TATA box

CAAT box: does 25% of genes transcribed in large quantities

TATA box: core region of the promoter

Additional Eukaryotic Regulatory Elements

Enhancer vs. Silencer vs Methylation

Methylation vs Acetylation of histones

• Can both be found both upstream and downstream

• Enhancer sequences: Activator binds it; speeds rate of transcription

• Silencer sequence: Repressor binds it; blocks transcription and prevents genes from being expressed

• Methylation and demethylation on cytosine+guanine (CpG islands): methylation blocks transcription and demethylation allows transcription

• Methylation of histone: blocks transcription so Heterochromatin formed

• Acetylation: makes histone negative , which makes DNA get off and loose → Euchromatin

Coding vs. Non-coding strands

Coding/ Sense strand: not used as template for transcription

Non-coding/Antisense strand: used as template

Transcription steps for Eukaryotes

1. RNA Polymerase II must become phosphorylated to be active

2. RNA Polymerase II binds to promoter of DNA and forms “closed complex”

3. RNA Polymerase II melts the DNA duplex near the transcription site and forms transcription bubble “open complex”

4. RNA Polymerase II catalyzes phosphodiester linkage of the first two rNTPs

5. Initiation factors are shed and elongation factors are recruited

6. RNA Polymerase II continues down non-coding DNA 3’-5’ and making a new strand that is growing 5’-3’ → Elongation

7. At transcription stop site, Polymerase releases DNA and RNA

Transcription steps for Prokaryotes

1. RNA pol holoenzyme binds to promoter of DNA, forming closed complex

2. Duplex is melted and transcription bubble “open complex” forms

3. Polymerase starts coding first few nucleotides

4. Sigma factor is released

5. Polymerase continues down non-coding DNA 3’-5’ and making a new strand that is growing 5’-3’ —>Elongation

6. Termination occurs when end gene is reached → Rho dependent or independent

7. Sigma factor rejoins and finds another promoter

Rho-dependent vs. Rho Independent

Rho independent: when termination sequence is reached, there is a self complementary AAA-UUU segment that forms a hairpin and stops transcription

Rho dependent: Once termination sequence is reached, ir recruits rho helicase, which hydrolyzes RNA away from DNA thus ending transcription

mRNA Post-transcriptional processing methods

3’ Poly A tail and 5’ cap —> their purpose is transport and stability

mRNA Splicing

• Components, how does it work

• Why do we need splicing

TQ: all of it

• Spliceosomes remove introns (noncoding) from sequence and leave the exons (coding)

• It’s a way for our cells to make more proteins from less genes; Gives us many alternative combinations

  • 3’ UTRs and 5’ UTRs , microRNAs —> Are there for regulation of gene expression

tRNA Processing

• 5’ sequence removed

• If there are introns, they are spliced out

• 3’UU is removed and replaced by CCA

• Base modification occurs as part of regulatory process ← TQ

What is a Copy number

It’s how many times the same gene can be transcribed and it varies depending on the cell’s needs