Cell Bio Review

March 15, 2010Cinderella Aquino

What are the differences between RNA and DNA?

• RNA is considerably smaller than DNA• RNA contains ribose instead of deoxyribose• RNA contains U instead of T• Differ from each other in function and special

structural modifications• RNA molecules can fold up into complex three

dimensional structures.• The 3-D shape of the RNA structure can confer function

RNA Polymerase produces a complementary RNA to the template strand

• The template strand of DNA serves as a template for RNA formation during transcription– Sometimes called the Antisense strand

• The coding strand is not directly involved in transcription.– Sometimes called the Sense strand– The coding strand sequence is extremely similar to the single-

stranded RNA molecule • Coding strand has T• RNA sequence has U instead of T

• Transcription is highly selective– Due in part to nucleotide sequence of DNA - signals to instruct RNA

polymerase where to start, how often to transcribe and where to stop

– Some regions of DNA, large number of transcripts are made, others none. 3

DNA is Transcribed By the Enzyme RNA Polymerase

• The RNA polymerase moves stepwise along the DNA unwinding the DNA helix at its active site

• RNA polymerases do NOT require primers to initiate transcription

• The template strand is read in the 3’ to 5’ direction

• The complementary RNA product is produced in the 5’ to 3’ direction

What are the different types of RNA?

• mRNA• tRNA• rRNA

• What do they do?• On what chromosomes is the genetic

info for rRNA found?

Comparison between Prokaryotes and Eukaryotes

• Prokaryotes: – Polycistronic– Operons– Bacterial protein synthesis proceeds on mRNA as it is being

synthesized• No nucleus!!• Transcription and translation can be coupled

• Eukaryotes:– Monocistronic– Eukaryotic primary transcripts are often processed into mature mRNA

– The processes of transcription and translation occur separately• Transcription occurs in the nucleus• Translation occurs in the cytoplasm

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Summary of the Prokaryotic Transcription Cycle

1. Holoenzyme assembles and binds to promoter

2. Polymerases unwinds DNA at site of transcriptional initiation

3. RNA synthesis begins4. After 10 nucleotides, sigma

factor is released5. Elongation of RNA occurs6. Synthesis of termination

signal7. Termination of

transcription

Rifampin and Actinomycin:Two Antibiotics that Inhibit Transcription

• Rifampin specifically inhibits initiation of RNA synthesis in BACTERIA

• Interferes with formation of 1st few phosphodiester bonds in RNA chain– semisynthetic derivative of rifamycins,

derived from strain of Streptomyces.– Used to treat TB (tuberculosis)

• Actinomycin D binds tightly to dsDNA, prevents it from being effective template for RNA synthesis in both pro and eukaroytes – Polypeptide antibiotic from different strain of

Streptomyces, – Used infrequently in treatment of various

malignant neoplasms: Wilm’s tumour, sarcomas.

– Adverse effects: bone marrow depression, GI toxicity; it is extremely irritating, produces severe tissue damage (toxic reactions frequent and severe)

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Eukaryotic RNA Polymerases

• RNA Polymerase I, II, and III carry out transcription in the eukaryotic nucleus– RNA Polymerase I: Precursor for 28S, 18S and 5.8S

rRNAs– RNA Polymerase II: Pre-mRNA, snRNA and microRNAs– RNA Polymerase III: Pre-tRNA, 5S rRNA

Preview of Eukaroytic Pol II Expression

• Several steps– Histone modification– Chromatin

remodeling– Addition regulators

• (enhancers, repressors promoters)

• Initiation complex binds with general and specific transcription factors

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Steps in RNA Pol II TF binding• TFIID binds to TATA box

• Can bind to DPE sequence in case of DPE-driven promoters

• TFIIA and TFIIB bind• RNA Polymerase II recruited to

promoter with TFIIF attached• Pol II Large Has a C terminal repeat

on C-terminal domain• CTD consists of 52 copies of a 7- amino acid repeat

• TYR SER PRO THR SER PRO SER

• CTD essential for cell viability• Phosphorylation State of CTD important

for Active

• Initiation complex completed by the addition of TFIIE and TFIIH

• TFIIH has both helicase and protein kinase activity

• Pol II phosphorylated by TFIIH, thereby releasing the polymerase to initiate RNA synthesis

Ribosomal RNA• rRNA participates in protein

synthesis as part of ribosome (RNA + protein complexes)

• Prokaryotes: 70S ribosome– Small subunit = 30S

• Small subunit composed of 16S rRNA and 21 proteins

– Large subunit = 50S • Large subunit composed of 23S + 5S

rRNA subunits, and 32 proteins

• Eukaryotes: 80S ribosome – Small subunit = 40S

• Small subunit composed of 18S rRNA and ~30 proteins

– Large subunit = 60S• Large subunit composed of 28S,

5.8S and 5S rRNA and ~ 50 proteins

rRNA Has Extensive Secondary Structure

• Final rRNA structure has variable intramolecular secondary structure (quite different than dsDNA)– Hairpins; double-helical stem and loop

structure where complementary base pairs hydrogen bond, More like A form dsDNA

– Provides structure to ribosome via 3-D interaction with proteins

• Antimicrobials such as erythromycin alter 70S prokaryotic ribosome. – One form of antibiotic resistance results

from bacterial methylation of rRNA by plasmid encoding methylase.

– Activation of methylase reduces ability of antibiotic to bind to ribosome

Secondary Structure of 16S

RNA

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RNA ProcessingrRNA

• Primary Transcript: an RNA molecule newly produced by transcription

• Ribosomal RNA processing involves cleavage of multiple rRNAs from a common precursor by ribonucleases – Transcribe spacers are

degraded. • Releases18S, 5.8S and 28S

– The pre-rRNA is also processed by methylation of 2’-hydroxyl group of the sugar ribose (small nucleolar RNA (snoRNAs play a role in process)

– Chemical modification to generate pseudouridines

Purple triangles = methylationRed dots = pseudouridines

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Transfer RNA processing

• Transfer RNA processing involves removal, addition, and chemical modification of nucleotides– Removal of 5’ leader

sequence, – excision of introns, – replacement of 3’ end with

CCA by nucleotidyltransferase

– Chemical modifications of bases • Methylation and deamination,

creation of unusual bases such as inosine, dihydrouracil, ribothymidine

Eukaryotic Processing to Mature Transcripts:

Begins with hnRNA: heteronuclear RNA1. 5’ cap: 7 methyl-guanosine added to 5’ end. Nuclear

guanylyltransferase adds 5’ guanosine while cytoplasmic guanine-7-methyltransferase adds methyl group to position 7 of base

2. Noncoding parts of gene cut out leaving behind exons (protein coding regions). Splicing– Introns = intervening sequences - noncoding regions are spliced

out– snRNA (small nuclear RNA or SnURPs) slice introns out, occurs in

spliceosome: alternative splicing: very important for protein diversity

3. Poly Adenosine (poly A) tail added by polyadenylate polymerase A after transcription . This poly A sequence is NOT translated

4. RNA Editing

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Splicing• Exons: sequences destined to appear in final mRNA• Introns: sequences within the primary transcript that do

not appear in mature, functional RNA– The number and sizes of introns varies greatly among genes

• Duchene Muscular Dystrophy: Dystrophin gene 2 million bp, 78 introns (introns account for 99% of the gene)

• Splice sites 5’-GU with 3’ AG– Splice Donor: GU at the 5’ boundary of the intron– Splice Acceptor: AG at the 3’ boundary of the intron– Branch Point: A

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Figure 6.47. Editing of apolipoprotein B mRNA In human liver, unedited mRNA is translated to yield a 4536-amino-acid protein called Apo-B100. In human intestine, however, the mRNA is edited by a base modification that changes a specific C to a U. This modification changes the codon for glutamine (CAA) to a termination codon (UAA), resulting in synthesis of a shorter protein (Apo-B48, consisting of only 2152 amino acids).