Lecture 4 Transcription

RNA Polymerase II Inhibitor

Alpha-amanitin(Amatoxins) a cyclic octa-peptide, produced by Amanita phalloides, also known as "death cap or destroying angel"--- Mushroom

Exons and Introns

Exons- coding, kept Introns- noncoding, spliced out. 90% of the pre- mRNA is removed as introns

5' Capping

Found on Heteronuclear RNA (hnRNA) Cap is 7-methylguanosine attached backwards to 5' terminal end. Needed to prevent degradation and as a signal for translation process Occurs pre-slicing

TFIIH

Helps in promoter melting with helicase Phosphorylates RNA polymerase II initiating the start of elongation , making it possible for RNA pol II to move downstream

TFIIE

Helps recruit TFIIH

Modifications of rRNA

rRNA synthesis and processing (snoRNA) takes places in nucleolus

Unusual Bases of tRNA

**Remember name and symbol only!** D = Dihydrouridine (pyrimidine) (methylated)- In the D loop Ψ = Pseudouridine (pyrimidine) (methylated)- In the TΨC loop I = Inosine (purine)

Initiation in Prokaryotes

1. RNA-pol recognizes the TTGACA region, and slides to the TATAAT region, then opens the DNA duplex. 2. The first nucleotide on RNA transcript is always purine triphosphate. 3. The pppGpN-OH structure remains on the RNA transcript until the RNA synthesis is completed. 4. The three molecules form a transcription initiation complex. No primer is needed for RNA synthesis. 5. The sigma subunit falls off from the RNA-pol once the first 3',5' phosphodiester bond is formed. 6. The core enzyme moves along the DNA template to enter the elongation phase. ++ Initiation Complex: RNA-pol - DNA-pppGpN- OH-3'

Sequence of TFs

1. TBP of TFIID binds to TATA 2. TFIIA and TFIIB 3.TFIIH and TFIIE --- after this step, the dsDNA opens and the RNA pol II attaches

Characteristics of mRNA

5' UTR (Untranslated Region) 3' UTR 5' Cap 3' poly-A tail Coding region

Post Transcriptional Modifications

5' cap, 3' poly A tail, splicing, and RNA edition

Similarities of transcription and replication

Both use DNA as the template. Phosphodiester bonds are formed in both cases. Both synthesis directions are from 5 ́ to 3 ́.

Ribozyme

Catalytic RNA that is able to self splice. Self-splicing occurs in Intron I and Intron II Forms a hammer head structure- conserves 13 nucleotides

Transcription

DNA --RNA

mRNA

Function: carries genetic information from nuclear DNA to the cytosol.Used as a template for protein synthesis.

tRNA

Function: transfer amino acid to a growing protein chain. Amino acid attachment site (A site) - covalently attached to the 3'OH end. Carries the anticodon to match the codon on the mRNA

TFIIB

Help in RNA pol II and recruits TFIIF, helps in start site selection

TFIIF

Help to RNA Pol in promoter binding

Prokaryotic and Eukaryotic Promoter Regions

In prokaryotes - the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site. The sequence at -10 is called the Pribnow box, and usually consists of the six nucleotides TATAAT In eukaryotes- TATA box, located 25 to 35 base pairs upstream of the transcription start site.

RNA

Joined by 3' to 5' phosphodiester bonds Contains ribose instead of deoxyribose Contains uracil instead of thymine. Can exist as single strands

alternative pre-mRNA processing

Key regulator of gene expression, lots of transcripts from a single coding gene! ---Seen in IgM gene

Initiation in Eukaryotes

Monocistronic - For each gene they have a regulatory sequence Promoter binds in between TATA and CAAT box. RNA- pol DOES NOT bind to the promoter directly, needs transcriptions factors (TF) first!

Alkaline Hydrolysis of RNA

RNA is labile to NaOH; while DNA is resistant. Requires a 2'OH - so RNA is sensitive but DNA is not. Great way to seperate DNA/RNA hybrids by removing RNA

RNA Polymerases in Eukaryotes

RNA polymerase I transcribes rRNA genes RNA polymerase II transcribes mRNA, miRNA, snRNA, and snoRNA genes RNA polymerase III transcribes tRNA and 5S rRNA genes. Requires cofactors to bind to DNA templates

Rifampicin

RNA-polymerase inhibitor a therapeutic drug for tuberculosis treatment, can bind specifically to the subunit of RNA-pol, and inhibit the RNA synthesis.

mRNA Splicing

Removal of noncoding regions--introns so that it is not translated into proteins. Takes place in the nucleus

Differences between transcription and replication

Replication --double strands, uses a primer, uses a DNA polymerase, A-T Transcription--single strands, does not use a primer, uses a RNA polymerase, A-U

Termination in Prokaryotes - Rho Dependent

Rho-dependent- Rho protein (helicase) attaches to the RUT site or Rho utilization site and moves towards the RNA polymerase using energy from ATP hydrolysis.

Termination in Prokaryotes - Rho Independent

Rho-independent -Reaches a termination signal (made of 30 -40 GC's nucleotides) The GC- region becomes a stem loop leading to the pause of the RNA-pol moving. The end region of U-A or poly U region is very weak and the hydrogen bonds break leading to disassociation of the polymerase. -Doesn't require ATP

Types of RNA

Ribosomal RNA (rRNA): functional components of the ribosome (prokaryotes: 70s, eukaryotes : 80s) Messenger RNA (mRNA): carry coding sequences from the DNA to the ribosome Transfer RNA (tRNA): deliver amino acids to the elongating protein Small Nuclear RNA(snRNAs) : splicing pf pre-mRNA Small nuclear ribonucleoproteins (snRNPs): splicing of pre-mRNA MicroRNAs (miRNAs) : interferes with mRNA and can decrease gene expression Small interfering RNAs (siRNAs): interfere with mRNA and decrease gene expression

Elongation in Eukaryotes

Similar to prokaryotic elongation, difference is that transcription and translation does not happen at the same time due to separation of nuclear membrane. Transcription- inside nucleus Translation- occurs in cytoplasm

What disease disrupts mRNA splicing?

Systemic lupus erythematosus- an often fatal inflammatory disease, results from an autoimmune response in which the patient produces antibodies against host proteins, including snRNP (small nuclear ribonuclear particles) Introns were kept in instead of being spliced out

TFIIA

TFIIA- Stabilizes TBP ( TATA Binding Protein) and TAFs ( TBP Associated Factors)

Transcription Factors

TFIID TFIIA TFIIB TFIIF TFIIE TFIIH

TFIID

TFIID- recognizes the promoter (TATA box)

Poly A Tail

Takes place in the nuclei At 3' end 40-200 Adenine nucleotides added Helps prevent degradation Once mature mRNA is inside the cytosol, the poly A tail gradually shortens. Occurs pre slicing

Termination in Eukaryotes

Termination sequence - AAUAAA followed by GU repeats. 1. Signal of the termination sequence activates endonuclease. 2. Post transcriptional modifications follow (poly A tail, etc.)

RNA polymerase in Prokaryotes

The enzyme responsible for the RNA synthesis is DNA-dependent RNA polymerase. Can bind to DNA template directly Contains 4 subunits : Alpha-Determine the DNA to be transcribed Beta- Catalyze polymerization Beta 1 - binds and opens DNA template Sigma- recognizes promoter initiation --Arranged in a quaternary structure

Elongation in Prokaryotes

The release of the sigma subunit causes the conformational change of the core enzyme. The core enzyme slides on the DNA template toward the 5' end. Free NTPs are added sequentially to the 3'-OH of the RNA strand. Transcription bubble: RNA-pol, DNA segment of ~40nt and the new RNA

Operon

Transcriptional region Polycistronic- one operon can transcribe several genes

Promoter

specific region of a gene where RNA polymerase can bind and begin transcription