Week 8 MolBio Flashcards

Eukaryotes = how many types of R. pol?

3. Pol I = makes pre-ribosomal RNA

Pol II: makes mRNA + noncoding RNA

Pol III: Transfer RNA (tRNA) + noncoding RNA + makes pre-ribosomal RNA

specific promoters + unique transcription factors

Yeast (Eukaryotic R. Pol II)

12 subunits: RPB1-RPB12

CTD = tail of 7 amino acids + in domain of RPB1

CTD: site of phosphorylation

CTD = key factor in transcriptional progression

CTD Transcription patterns

different positions of CTD are phosphorylated by other kinases based on the stage of transcription and this difference in positioning helps cellular machinery to call specific items needed for that phase

How do promoters bind to R. Pol II

promoters on DNA bind to R. pol II w/help of trancription factor protein TBP (TATA box- Binding Protein).

TBP binds to TATA box bending DNA so R. pol can swoop in and make a transcription bubble

Assembly of pre-initiation complex

A bunch of transcription factor proteins help recruit R. pol II to bind to DNA

Transcription Factor Proteins

1. TBP: binds to TATA box

2. TFIIA: stab bind TFIIB + TBP to promoter

3. TFIIB: binds to TBP, ‘recruits’ it to Pol II-TFIIF complex

4. TFIID: NEEDED to start initiation if prom = no TATA box

5. TFIIE: recruits TFIIF

6. TFIIF: binds tightly to Pol II, binds to TFIIB + stops binding of Pol II to nonspecific DNA seq

7. TFIIH: unwinds DNA at promoter (helicase activity), phosphorylates Pol II (at CTD), recruits nucleotide-excision repair proteins

Initiation

• Helicase activity of TFIIH facilitates unwinding of DNA, forming transcription bubble

• CTD is phosphorylated on serine residues by kinase domain of TFIIH and CDKs

• TFIIH and TFIIE dissociate

• RNA Pol II clears the promoter

Elongation and Termination

RNA Processing

a

Transcription and Transcription in Eukaryotes

NOT COUPLED. SEPARATE. Transcription happens in Nucleus and Translation happens in Rough ER/Ribosomes. (thats why mRNA is processed to prevent degradtion)

5’ Cap details

What the 5' Cap is: A 7-methylguanosine group attached to the 5' end of the RNA via a unique 5',5'-triphosphate linkage.

The CTD Connection: The cap-synthesizing complex doesn't just float around—it hitches a ride directly on the phosphorylated CTD tail of RNA Polymerase II so capping can happen co-transcriptionally right as the mRNA emerges.

Addition of the Poly(A) tail

a

Polyadenylation factors

AAUAAA: polyadenylation/cleavage signal in mRNA sequence

Polyadenylation factors: Recognize and bind the cleavage signal

Endonuclease: Cleaves mRNA 10-30 nucleotides downstream of AAUAAA

Polyadenylate Polymerase: Synthesizes polyA tail (80-250 nucleotides) starting at cleavage site

Introns and Exons

Introns: unimportant non-coding portions of transcript

Exons: coding portions of transcript

Introns cleaved after capping while polyadenylation happens

Ovalbumin gene = 7700 bp is 1872 nucleotides after processing w/cap

Mechanism of splicing

Transesterification Reaction

Group I Intron Splicing Mechanism

Group II Intron Splicing Mechanism

What is spliceosome?

A spliceosome is a ribonucleoprotein made up of both RNA and protein molecules

Splicesomal Splicing mechanism, Part I

Splicesomal Splicing mechanism, Part II

Why do we need introns?

Alternative splicing: basically, the exons can be rearranged in different orders which leads to different isoforms of the same protein from the transcript = greater genetic diversity

5 protein classes needed to activate transcription

1. Activators: bind to enhancer sequence(s)

2. Architectural regulators: allow looping of DNA to facilitate interactions between proteins bound to sequences that are far away

3. Co-activator: intermediaries between activator and Pol II complex

4. Basal transcription factors - TFII proteins for Pol II

5. Chromatin Remodeling Complexes (not shown)