Transcription and RNA Processing 1

RNA Polymerase I How it transcribes rRNA genes

1. 150 bp promoter recognized by transcription factors. 2. Recruitment of RNApol1 3. Large 45S pre-rRNA processed to 28S, 18S, and 5.8S rRNAs

tRNA processing

1. Begins as long precursor (pre-tRNA) 2. Cleavage: 5' end by ribozyme RNase P 3' end by typical protein RNase 3. CCA terminus added to the 3' end - site of amino acid attachment - Some tRNA and rRNA genes have introns that are removed by splicing - Modification of bases: play important role in protein synthesis by altering base-pairing properties of tRNA molecules and stabilize hairpin loops.

What are the two models of initiation of transcription by RNApol2?

1. Stepwise = binding to GTFs, pol 2, and Mediator 2. Holoenzyme = Binding of a single multiprotein complex composed of pol 2, Med, and the GTFs.

RNA Polymerase II Mechanism

1. TFIID (including TBP) binds promoter. 2. TFIIB binds BRE and TBP 3. TFIIF binds to RNApol2 4. TFIID/TFIIB/DNA complex binds the TFIIF/RNApol2 complex 5. TFIIE and TFIIH bind the complex 6. Mediator binds to the CTD of RNApol2 and various GTFs and factors specific to promoter. 7. Distinct subunits of TFIIH unwind DNA and phosphorylate the CTD of RNAPol2 8. Phosphorylation triggers loss of affinity with pre-initiation complex and the mediator proteins (RNApol2 and mediator proteins released) and triggers the beginning of the transcription.

What do each of the eukaryotic rRNA's form?

18S -> 40S ribosome 28S, 5.85S, 5S -> 60 S ribosome

What rRNAs do bacteria have? How derived?

23S, 16S, 5S Derived by cleavage of a single transcript.

Which rRNAs are derived by a single transcript?

28S, 18S, 5.85S

What species of rRNAs do eukaryotes have?

28S, 18S, 5.85S, 5S

Where are rRNA genes located in human genome?

28S, 18S, 5.8S -> Chromosome 13, 14, 15, 21, 22 5S -> Chromosome 1

What rRNA is transcribed outside of the nucleolus?

5S rRNA

Do cells need many ribosomes?

Cells require many ribosomes to meet their needs for protein synthesis. Actively growing cells contain 5-10 mil ribosomes that must be synthesized eat time the cell divides.

Characteristics of Eukaryotic RNA Polymerases

Each produces a different class of RNA Require general transcription factors to initiate transcription. 9 conserved subunits, 5 related to bacterial RNA polymerase subunits. Don't require primer - read template strand 3'-5' direction

Transcription in eukaryotes Where is eukaryotic DNA contained? How many RNA polymerases? Where does transcription occur?

Eukaryotic DNA is contained within nucleus. Eukaryotes have 3 RNA polymerases Eukaryotic RNA polymerases require many additional proteins to transcribe RNA. Eukaryotic transcription occurs on chromatin, which is wound tightly and makes it difficult for RNA polymerase to access the DNA. Eukaryotes - mRNA is synthesized in the nucleus first the transported to cytoplasm before protein synthesis but prokaryotes begin translation while transcription is till in progress.

Where is most rRNA transcribed and processed?

Nucleolar organizing regions which organize chromosomal regions that contain the genes for 5.8S, 18S, and 28S rRNAs.

What is the pre-initiation complex comped of?

RNApol2 + 5 general transcription factors are minimally required for initiation in vitro.

How is rRNA processed?

Similar in prokaryotes and eukaryotes Successive cleavages of pre-rRNA transcript.

Eukaryotic rRNA processing SnoRNA

SnoRNA function in prerRNA processing: - Adding methyl groups to ribose residues and bases - Converting uridine to pseudouridines

What does the mediator do?

Stimulates basal transcription Plays role in linking GTF to the gene-specific transcription factors that regulate gene expression

RNA Polymerase I What does it synthesize?

Synthesize 5.8S rRNA 18S rRNA 28S rRNA.

RNA Polymerase II What does it synthesize?

Synthesize: mRNA miRNA some snRNA some scRNA

What is TAF and what does it bind in RNAPol2 initiation complex?

TBP associated factors Binds to downstream elements

RNA Polymerase II Promoters

TF2B recognition site TATAA box - Located 25-30 nucleotides upstream of transcription site = In 10-20% of RNA polymerase II promoters Initiator element Downstream elements

How does RNApol2-TFIIF join the TFIID/TFIIB/DNA complex?

TFIIB Acts as a bridge to join RNApol2-TFIIF to the complex.

RNA Polymerase III Where are the promoters?

The promoters for 5s rRNA and tRNA are unusually located downstream. Promoter for snRNAs is located upstream.

What is a ribozyme?

an enzyme in which RNA rather than protein is responsible for catalytic activity; RNA is active without protein.

TFIIH

contains helicase and protein kinase. Phosphorylates Serine-5 in CTD repeats of RNAPol2.

What are snoRNPs

small nuclear RNAs complex with several proteins that function in pre-rRNA processing.

What are snRNAs?

small nuclear RNAs involved in splicing and protein transport.

RNA Polymerase III What does it synthesize?

synthesize : tRNA 5S rRNA some snRNA some scRNA