L07 Chem 481 Lecture Notes - Lecture 14: Rna Polymerase Iii, Nucleoplasm, Rna-Dependent Rna Polymerase
13 Dec 2016
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30 September 2016
Lecture 14: Eukaryotic Transcription
I. Eukaryotic Transcription
A. RNA Polymerases
1. Eukaryotic RNA Polymerases
a. RNA pol I, multi-subunit, produces rRNA and tRNA in the nucleolus
• rRNA production is the major transcriptional activity in cells, ~80% of the activity
• Major ribosomal RNA genes
b. RNA pol II, multi-subunit, produces mRNA, miRNA in nucleoplasm
• Protein encoding genes and few small RNA genes (nascent transcript)
c. RNA pol III, multi-subunit, produces tRNA, 5s rRNA, ncRNA in nucleoplasm
• Transcribes small ncRNA genes and tRNAs
d. mtRNA pol is a single polypeptide (DNA pol I related), transcribes all mtDNA genes in
the mitochondrion
• Not like other RNA pol, more like a DNA replicase
e. Plants have two additional nuclear RNA pol (Pol IV and Pol V) to generate ncRNAs that
are required for transcriptional gene silencing via the RNA-directed DNA methylation
pathway – Pol II has similar function in animals
f. The 3 types of nuclear RNA pol in eukaryotes each require additional protein factors to
initiate transcription
2. E. Coli Processing of RNA
a. The nascent transcript folds on itself as it is produced, it is impossible to disentangle
• Folding, assembly, and modification all occur during production of the RNA
b. In E. Coli will often have ribosomal gene repeats (visualized from fixed preparation in
grey image) with ribosomal parts and tRNAs that will be cut from the nascent transcript
• After maturations, the pieces in between are cut out releasing the rRNAs and tRNAs
3. Pol I- Eukaryotic rRNA Processing in the Nucleolus
a. Lots of processing is necessary for optimizing the efficiency of function of the RNAs
b. Lots of machinery is required to make changes once RNA pol I transcribes,
cotranscriptional modifications and cleavages (irreversible)
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• Involves about 200 proteins (many enzymes), 200 snoRNAs and snoRNPs, about 100
nt are 2`-O-methylated in riboses, ~95 uridines modified to pseudouridines, ~10 bases
methylated
• Order and location (cytoplasm vs. nucleoplasm) of cuts differ between lower and
higher ekaryotes
c. Transcript is completely described but different spacers and sites are cut by different
machinery in processing
d. Pol I also is involved in transcription and processing of larger rRNAs in mammals
• As in all eukaryotes, the rDNA genes exist in tandem copies, separated by non-
transcribed spacers
4. Ribosome Biogenesis
a. The rDNA transcription, pre-rRNA modification and processing occurs simultaneously
(picture on left can be viewed as a process or snapshot)
b. Initial transcription factors (TFs) bring in a bunch of other factors
c. snoRNPs (small nuclear ribonuclear particles) contain partly folded and party open
strands, can interact with sequences
d. Pol I is associated with a large number of processing factors at the promoter region,
forming a huge Pol I holoenzyme
e. After initiation of transcription, numerous pre-rRNA processing factors, like modification
guide snoRNPs and the transcription associated U3 snoRNA proteins (t-Utp) are
recruited
• Pre-rRNA modification occurs before assemply of the small ribosomal subunit
processome
• The t-Utp complex binds the pre-rRNA very early, promoting the assembly of
additional pre-rRNA processing factors including U3 snoRNP
f. RNA folded into final form and modified as ribosomal proteins bond during transcription
and then cleavage occurs at the end
• Many proteins and factors are associated with the RNA
g. When elongating, Pol I reaches the 3` ETS, the RNAase III endonuclease, Rnt1, is loaded
onto the pre-rRNA and cleavage in the 3` ETS proceeds cotranscriptionally
• Termination usually by protein “road blocks” stalling to allow Rnt1 to cleave the
transcript
h. Most higher eukaryotes don’t have introns in ribosomal RNA genes (increases efficiency)
5. Pol III Transcription of 5s rRNA and Most tRNAs
find more resources at oneclass.com
find more resources at oneclass.com
