L15 Transcription And Rna Processing

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rRNA

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Ribosomal RNA
structural component of ribosomes and serves as a framework for the association of ribosomal proteins
80% of total RNA

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tRNA

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Transport RNA
transports amino acids to ribosome and ensures their alignment prior to peptide bond formation
15% of total RNA

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mRNA

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Messenger RNA
serves as template for synthesis of protein in eukaryotes, carries information specified by DNA to cellular protein synthetic machinery
5% of total RNA

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hnRNA

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Heterogeneous Nuclear RNA
precursors to mRNA

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SnRNA

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Small Nuclear RNA
structural and regulatory RNA's that are components of spliceosome, often required for splicing of eukaryotic mRNAs

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ScRNA

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Small Cytoplasmic RNA
involved in cellular mechanism whereby proteins destined for secretion, insertion into membranes or inclusion in lysosomes are sequestered inside of the endoplasmic reticulum

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What drives RNA synthesis?

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hydrolysis of PPi

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alpha subunit

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subunit of DNA-dependent RNA Polymerase for Prokaryotic cells
2 of them
Function: chain initiation

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beta subunit

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subunit of DNA-dependent RNA Polymerase for Prokaryotic cells
1 of them
function: chain initiation and elongation

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beta prime subunit

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subunit of DNA-dependent RNA Polymerase for Prokaryotic cells
1 of them
function: DNA binding

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sigma subunit

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subunit of DNA-dependent RNA Polymerase for Prokaryotic cells
1 of them
function: promoter recognition

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omega subunit

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subunit of DNA-dependent RNA Polymerase for Prokaryotic cells
1 of them
function: unknown

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Which one is more error prone, DNA or RNA?

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RNA

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What is the holoenzyme and what subunits does it consist of?

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initiation portion of prokaryotic RNA polymerase
2 alpha, beta, beta prime, and sigma

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What is the core enzyme and what subunits does it consist of?

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elongation portion of prokaryotic RNA polymerase
2 alpha, beta, beta prime
NO SIGMA

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Where does the sigma subunit bind?

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to the promoter on the coding strand

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What are the two parts of the prokaryotic promoter?

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-10 sequence (PRIBNOW Box)
-35 sequence

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What is the consensus sequence of the -35 sequence?

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TTGACA

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What is the consensus sequence of the -10 sequence?

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TATAAT

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What is another name for the coding strand?

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sense (+) strand

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What is another name for the template strand?

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antisense (-) strand

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List and explain prokaryotic transcription termination methods.

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Factor-independent - template contains two symmetrical GC-rich areas and a poly-A area that creates a stem-loop-stem and down stream poly-U sequence on the RNA strand
rho factor dependent- rho factor is an ATP-dependent DNA:RNA helicase

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Polymerase I

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Eukaryotic RNA Polymerase
location: nucleus(nucleolus)
synthesizes pre-rRNA
a-amanitin:insensitive

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Polymerase II

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Eukaryotic RNA Polymerase
location: nucleus
synthesizes pre-mRNA and some SnRNA
a-amanitin; V-sensitive

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Polymerase III

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Eukaryotic RNA Polymerase
location: nucleus
synthesizes pre-tRNA, 5S rRNA, SnRNA
a-amanitin: sensitive

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Mitochondrial Polymerase

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Eukaryotic RNA Polymerase
location: mitochondrion
synthesizes mitochondrial RNA
a-amanitin: insensitive, inhibited by rifampicin

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What are three common components of the eukaryotic promoter?

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TATA box (aka HOGNESS)
CAAT box
GC box

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What are the consensus sequences of TATA box, CAAT box, and GC box?

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TATA- TATA, about -25 to -35bp from start point
CAAT box- CCAAT, variable (-40 to -100)
GC box- GGGCG, variable (-40 to -100)

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What are two other eukaryotic promoter elements?

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INR- initiator
CpG islands- GC-rich areas 20-50 nucleotides long, found on genes that are transcribed at low rates (house keeping genes)

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How does polymerase I attach to DNA and what does it synthesize?

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it attaches using an essential core element and and upstream element.
it synthesizes multiple rRNAs, but not 5S

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How does polymerase III attach to DNA and what does it synthesize?

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transcribes tRNA and 5S rRNA
Polymerase III binds using three transcription factors that bind to promoter region inside the gene
rRNA gene- TFIIIC and TFIIIA bind to Cbox, and TFIIIB binds to gene
tRNA gene- TFIIIC binds to Abox and Bbox and TFIIIB binds to gene

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What are the four steps of post-transcriptional tRNA processing?

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1. 5' end cleaved with RNaseP(ribozyme) and 3' end cleaved with RNaseD
2. CCA added to 3' end by tRNA nucleotidyltransferase
3. base modification
4. for euk tRNA, 14-ntd intron spliced out

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What steps are involved in eukaryotic mRNA processing?

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1. pre-RNA has a 7-methylguanosine cap added by a 5'-5' triphosphate linkage
2. 3' polyadenylation
3. splicing of introns
takes place in nucleus and is very rapid

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What steps are involved in 3' polyadenylation?

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1. Complex of endonuclease and polyadenylate polymerase binds to signal sequence
2. RNA is cleaved 11-30 nucleotides 3’ to AAUAAA.
3. Polyadenylate polymerase synthesizes poly (A) tail of 20-250 nucleotides beginning at cleavage site

driving force is cleavage of ATP to PPi

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Rifampicin

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inhibits initiation of RNA synthesis in prokaryotes and mitochondria
binds to the beta subunit of bacterial RNA polymerase preventing formation of first phosphodiester bond

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alpa-amanitin

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toxic mushroom poison
binds eukaryotic RNA polymerase II preventing formation of eukaryotic mRNA precursors (hmRNAs) in elongation

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Actinomycin D

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from Streptomyces
binds to double-stranded DNA, intercalates at G-C bp preventing movement of RNA polymerase
inhibits RNA synth, not replication or protein synth
not specific to eukaryotes or prokaryotes

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Azidothymidine

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aka AZT
inhibits reverse transcriptase
chain terminator of DNA synth