Chapter 26: RNA Metabolism

Three parts of RNA Metabolism

1. Transcription

2. Ribozymes

3. Processing

Transcription Reaction

Ribose nucleoside triphosphates are added to 3' end of mRNA via using -OH to attack Pi

RNA Polymerase

Catalyzes mRNA synthesis using Magnesium

Promoter

Sequences RNAP bind to to start transcription

No primer is needed

Supercoiling due to transcription

Overwinding upstream

Handled by Topiosomerases

Template Strand

Used by RNAP to make mRNA

Complementary to mRNA

Coding Strand

nontemplate strand

same sequences as RNA

Regulatory sequences contained here

Can only one stand of a chromosome be the template?

Both strands can have either role for dfferent genes

RNA Polymerase holoenzyme structure

two alpha subunits

Two beta

1 omega

1 sigma

Sigma subunit

directs RNAP to promoter

Most common sigma

Sigma 70

RNAP and 3'--->5' exonucleases

No exonucleases or proofreading

Not needed because mRNA has a short lifespan

Bacterial Promoter

TATA Sequences and UP element

TATA Sequences

-10 and -35 AT sequences that sigma binds to

UP element

AT sequence that binds to the alpha subunit

Why does TATA and UP have so many A-T's?

Makes strand seperation easier

Footprinting Technique

Determine the sequence where a protein binds DNA

Footprinting procedure

1. Isolate DNA

2. Radiolabel DNA

3. Bind proteins to 1 DNA sample and have another DNA sample with no proteins

4. Treat both with a DNA cleaving enzyme or chemical

5. Run a gel

5. Isolate samples from bands on control but not cleaved and sequence them

Bacterial Transcription Process (5 Steps)

1. RNAP binds promoter using sigma to form closed complex

2. Open complex forms

3. Promoter Clearance

4. NusA displaces Sigma 70 and elongation occurs

5. NusA dissociates and termination occurs

Sigma 32

Used to bind RNAP to heat shock promoters, only used under high heat

Sigma 70

housekeeping sigma factor

attracts RNAP to housekeeping promoters

How do bacteria regulate transcription?

Regulate affinity of RNAP for a promoter

Different promoter sequences for different genes

Use activator and repressor proteins

Rho-independent Termination

3' end of template strand has lots of A's

Leads to lots of U's which forms a self-complementary hairpin in RNA to end transcription

Rho-dependent Termination

Rho binds rut site and seperates RNA and DNA using ATP-dependent helicase activity

RNAP I

makes rRNA precurosors

RNAP II

makes mRNA

RNAP III

makes tRNA and small RNA's

RNAP IV

used in plants to make siRNA

Mitochondrial RNAP

transcribes genes located on the mitochondrial chromosome

3 Parts of eukaryotic promoters

1. TATA at -30

2. Inr (initiator) sequence at +1

3. Regulator elements upstream

RNAP II structure

12 subunits

Carboxy terminus with repetitive elements

4 Steps of Eukaryotic Transcription

1. Assembly: RNAP and TFII's bind promoter to form preinitiation complex

2. Initiation: TFIIH opens DNA and phosphorylated CTD of RNAP to displace all TFII's

3. Elongation: elongation factors bind, processivity increased and phosphorylation on CTD altered

4. Termination: CTD is dephosphorylated, RNAP stops and mRNA is released

Main way that stage of transcription is controlled?

Phosphorylation of CTD of RNAP

TFIIH's role in DNA repair

TFIIH recruits nucleotide-excision repair system to DNA lesions

actively transcribed genes are repaired more often

xeroderma pigmentosum

mutation in TFIIH, cannot recruit NER system to repair pyrimidine dimers

Results in damage from sunlight

Actinomycin D and acridine

antibiotic

intercalate bacterial DNA to block RNAP

Rifampicin

binds beta subunit of bacterial RNAP

prevents promoter clearance

alpha-amanitin

fungal toxin

blocks Pol II and III of animals

Primary Transcript

mRNA before it is processed to a mature transcript

When does mRNA processing occur

during transcription

modification proteins bind to CTD of RNAP

5' cap

7-methylguanosine made from GTP and added via 5', 5' triphosphate linkage

Used to protect mRNA from nucleases and provide a ribosome binding site

Methylation in 5' cap

2' OH of two nucleotides after the cap are methylated using SAM

4 classes of introns

Type I and II

Spliceosome

tRNA

Group I and II Introns

self-splicing, found in bacteria, mitochondrial, chloroplast, nuclear DNA

Spliceosomal introns

most common, found in protein coding genes

tRNA introns

spliced by endonucleases and joined bakc by ligases

Group I intron splicing

The -OH from a free GMP or GDP is used as a nucleophile to attack the PD bond between a U and A at the end of the first exon

3' of the U is then used to attack the 5' of the other exon

Group II intron splicing

2' OH of A in intron attacks PD bond between U and intron

forms laviat-like intermediate with 2',5' bond

free OH from first exon attacks nucleotide at 5' of second exon to release intermediate

snRNPs

small nuclear ribonucleoproteins, used to make spliceosome

snRNA

small nuclear RNA

U1, U2, U4, U5, U6

U1

Binds GU at 5' end intron

U2

Binds AU at 3' end of intron, cause bulge to make a second A near the 3' end a good nucleophile to create a lariat-like intermediate

U4, U5, and U6

binds mRNA to attract 50+ other proteins to form spliceosome

Poly A Tail

Used to protect mRNA and provide a binding site

mRNA Poly A Tail Process

1. RNA is synthesized beyong a cleavage sequence

2. Endonuclease cleaves mRNA to AAUAA sequence

3. Polyadenylate Polymerase adds A to 3' end

2 Ways to use Processing to Increase Protein Diversity

1. Alternate splicing: remove different exons

2. Use different Poly-A Tail sites

Why to tRNA and rRNA need bases besides the normal 4?

allows for alternative hydrogen bonding and specialized 3-D folding

Bacterial Ribosomal RNA Creation

1. 30S RNA is transcribed and modified

2. Cleavage into subunits

3. Nucleases cleave excess

4. End with tRNA, 16S, 23S, and 5S rRNA

tRNA Synthesis

1. Base Modification and Cleavage

2. Splicing

RNase P

A ribozyme that cleaves the 5' end of pre- tRNAs.

RNase D

Cuts 3' end of tRNA

miRNA

regulate translation by cause mRNA cleavage or preventing translation

miRNA synthesis process

1. pri-miRNA made in nucleus

2. Drosha and DGCR8 cleave to pre-miRNA

3. Ran and Exportin move precursor to cytoplasm

4. Dicer converts to dsRNA

5. Helicase removes complement strand

6. miRNA loaded onto RISC to target mRNA

Ribozymes

RNA molecules that cleave themselves or other RNAs

Need 3D structure to function

How are ribozymes like RNA's (4)

1. Saturable

2. Active Sites

3. Km

4. Competitive Inhibitors

Ribozyme mechanism

Use 3' OH to attack 5' Pi (transesterification) and PD bond hydrolysis

3 Examples of Ribozymes

1. Self-splicing introns

2. hammerhead

3. RNase P

mRNA degredation

varied to regulate transcription

hours in eukaryotes

Steady State mRNA

rates of synthesis and degrdeation are equal

Ribonucleases

enzymes that degrade RNA

Retrovirus life cycle

enter cell

use RT to make RNA to DNA

degrade RNA and make second DNA strand

DNA incorporated into host DNA using integrase

Reproduce using host machinery

gag

encodes long polypeptide cleaved into smaller proteins for viral core

pol

encodes protease to cleave gag polypeptide

env

encodes envelope

LTR

long terminal repeat

used to integrate DNA

Three Functions of RT

1. RNA dependent DNA Synthesis

2. RNA degredation

3. DNA-dependent DNA Synthesis

3 RT features

1. Zn 2+

2. tRNA primers

3. no 3' to 5' proofreading to cause rapid viral evolution

Src

Rous sarcoma virus has this gene

Encodes tyrosine kinase which causes sarcomas by stimulating cell division

HIV

retrovirus, kills T lymphocytes to cause AIDS

mutates rapidly, at least one difference per RNA

RT Inhibitors

Analog nucleotides and nucleosides

ends in dine or sine

Protease inhibitors

Inhibit cleavage of proteins to form new viruses

end with avir

Retrotransposons

transposons that encode RT homolog

move between chromosomes using RNA intermediates

Why can telomeres not be easily replicated by DNAP

no primers are avaliable

Telomerase Function Steps

1. Telomerase binds 3' end of DNA

2. Telomerase translocates so that 5' strand has overhang using CA repeats

3. Telomerase adds in GT repeats on 3' end

4. Telomerase dissociates, DNAP fills in gap and RNA removed with RNase

5. binding proteins bind the telomere to protect it