8. RNA Processing Part 4: tRNA Transcription and Processing

The proposed splicing endonuclease catalysis has what AA?

Lysine Histidine and Tyrosine

reexport into the cytoplasm with what?

Msn5, Los1

what happens from then?

Mtr10 and back out into the cytoplasm

2' Phosphotransferase uses _____________ cofactor to pop off phosphate

NAD+

RNA pol III binds _______________ the transcription area. (normally)

On the transcription area. Not before it like in pol II

tRNA is exported into the cytoplasm and what happens?

Splicing modifications charging

are tRNA processed?

Yes

Kinase does what?

adds phosphate to 5'OH

what is created from splicing out?

anti-codon

TFIIIC binds where?

around A and B

2'-3' cyclic phospho diesterase does what?

cleaves phosphate bond from 3'OH and phosphate to makes a 2' P

What is special about the enzyme that processes 5' end?

composed of RNA and protein (has RNA***RNA world hypothesis ) RNA is a ribozyme (catalyzes the cleavage on 5' end)

the promoter in RNA pol III is ______________________ from RNA pol II

different

The 3' end of tRNA can be processed by _________ and ________s

endos and exos nucleases

How can CCA at the 3' end of the tRNA be added by the enzyme?

enzyme must be able to bind CTP or ATP

what gets spliced out?

intron in the anti-codon area on intermediate tRNA

What is wrong with pKa 10?

its far from physiological pH

Ruler mechanism does what?

measure the distance between nucleotides to cleave in the proper spot

tRNA intron processing: are tRNA introns removed by the same mechanism as mRNA introns?

no

5' processing occurs in the?

nucleolous

Transcription occurs in the?

nucleolus

3' processing CCA addition Nuceloplasmic modifications modifications at INM occur where?

nucleus

Charged, spliced and modified tRNA go into what next?

nucleus

what kind of processing?

precursors are processed at 5' and 3' ends. Introns on the tRNA anticodon area are spliced out

tRNA introns are the only ones removed by:

protein enzymes

30 types in _____________

rRNA

RNase P is a __________________

ribozyme (has catalytic component)

Endonuclease has to be very___________ on the tRNA splicing

specific

what do these AA do?

stabilize the phosphate intermediate

TFIIIB binds where on DNA?

start of transcription: transcription start site (TSS)

81 different types in ___________________

tRNA

type 2

tRNA

when you see a 2'-3' cyclic phosphate, you know what?

that the 2'OH is the nucleophile

bacteria can do what during transcription?

translation

RNA pol II binds _____________ before the transcription area?

upstream

what is the nucelophile??

water

what is the pKa of Tyrosine OH?

~10

2' Phosphate needs to leave now... how?

2' Phosphotransferase

you also create a _______________________________ after cleavage

2'-3' cyclic phosphate (on 5' end)

how many different types of RNA poly III promoters are there?

3 Type 1 type 2 type 3

CCA is added on what end?

3' end

types of modified bases in tRNA and rRNA

4-Thiouridine (S4U) Inosine (I) 1-methylguanosine (m1G) N6-isopentenyladeonosine (i6A) Ribothymidine (T) Pseudouridine (psy (greek)) Dihydrouridine (D)

most often you create a _____' phosphate and a _____' OH

5' phosphate 3' OH

type 1

5s

tRNA modifications: how many known modifications are there?

96 known

tRNA Primary transcripts need to do what to make Mature tRNA?

Cuts on 5' and 3' ends Adds CCA base modification splicing

type 3

His U6

RNase A has what AA in the active site?

His and Lys His 119 His12 Lys 41

CCA polymerase has how many active sites?

1 active site

Ligase has 3 activites:

1) 2'-3' cyclic phospho diesterase 2)Kinase 3)Ligation

Models:

1) 3 active sites 2) 1 active sites

tRNA ligase will use the same mechanism as DNA ligase which is:

1) Enzyme uses lysine (NH3+) to an ATP to make Enzyme---NH2-- ribose---A 2) this 5' phosphate will bind Enz-AMP 3) 3'OH nucleophilic attack on 5' phosphate to seal NICK

Why must 2' phosphate be removed?

1) could interfere w/ downstream events 2) changing tRNA w/ alpha alpha 3)translation -EF-Tu -Ribosome -mRNA

if the tRNA ligase mechanism is similar to what we learned about DNA ligase, what steps must have to happen a the nick before ligase can even start?

1) have 3'oh instead of 2'-3' cyclicphosphate 2) have a 5' phosphate

Yeast ligase will:

1) take 3' cylcic phosphate and free up 3'Oh by cleaving the bond to phosphate 2) uses Kinase on 5' end 3) now it can do DNA ligase mechanism (tRNA) Activates the 5' P with AMP

Ligation (3 steps)

1. AMP on lysine (uses ATP) 2. Attach AMP to 5' Phosphate 3. catalyze attack of 3'O on 5' Phosphate

tRNA splicing endo makes how many cleavages?

2

What could lower it to a useful range in the enzyme (pKa ~7)

Put a plus charge near it (Arginine)

what transcribes tRNA genes in Euk.?

RNA pol III

What transcribes tRNA genes in Bacteria?

RNAP

Endonuclease is similar to ____________ A.

RNase A (first RNase to be understood)

Cuts: 5'--------primary transcript-------3' what cuts on the 5' end (between primary transcript and 3' end?

RNase D

Cuts: 5'--------primary transcript-------3' What cuts on 3' end (between 5' and primary transcript tRNA)

RNase P