bio 108 lec 6b
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Transcription
RNA polymerase molecules on DNA are
producing ribosomal RNA moleculesthat bind to ribosomal proteins.
Transcription starts at a "promoter."Transcription ends at a "terminator."
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A transcription unit = a gene or operon
Genes are tightly packed (some
overlapped) in bacterial genomes butloosely packed with many long intergenic
spaces in eukaryotic genomes.
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Bubble
Bubble
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Transcription cycleR Y R
+1
Promoter for E. coli 70-RPase
Holoenzyme (core + )
finds and binds a
promoter.
A region around -10
and +1 is unwound
to form a bubble.
interacts with the -35
and -10 hexamers.
Isomerization from
closed
to open binary complex
Nucleotides bind to form
ternary complex
Abortiveinitiation
cycling
Closecomplex
Open
complex
Elongation
complex
Termination
complex
Short RNAs are produced, as
the contact with -35 is lost.
Short RNAs are repetitively releasedand new RNAs are produced.
is released
and promoter
is cleared.
Elongation is
processive
and stable.
RNA is released
in termination
and RPase
dissociates
from DNA
Intrinsic terminators code for
RNA hairpin - oligo(U)
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Eukaryotic transcription
A RPase II promoter
Nuclear RPase I, II, III and IV (?),
Mitochondrial and chloroplast RPases
Initiation complex
TFIID binds and
bends TATA box.
Many initiation
factors are
loaded.
by TFIIH during
Abortive cycling
TFIID remains at
promoter and PRPasemoves down.
Many proteins (>100 subunits)
must assemble around the +1site.
The order of assembly varies
from gene to gene. Some
protein complexes are pre-
assembled before brought toDNA together.
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Eukaryotic mRNA processing in nucleus5'-UTR 3'-UTR
Exons + Introns
All the mRNA
processing events,
capping, splicing,
3'-end processing
occur during
transcription within
nucleus.
The processing
factors bind to CTDof RPase and act on
the mRNA parts
that are coming out
of the RPase.
Only RPase II hasCT
Capping at the 5' end of mRNA
5'-P-5'
linkage CBC (cap
bindingcomplex)
marks a
successfully
capped end.
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Eukaryotic mRNA splicingTwo sequential Exon = Expressed part, Intron = Intervening part
transesterification
reactions at a site
EJC (exon junction
complex) marks asuccessfully spliced site
5'-Splice
site
Branch
site
3'-Splice
site
The 5'-splice, branch and
3'-splice sites are highly
homologous in all splicings.
Splicing occurs sequentially
as soon as CTD-boundsplicing factors recognize
relevant sites sequentially
coming out of RPase II.Thus, a mature RNA of
exon3-exon1-exon2 cannot
be made in vivo.
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Spliceosome: snRNA-snRNA interactions
SRproteins
mark
exons.
RNA-RNA
Rearrange-
ments
using ATP
hydrolysis.
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Alternative and abnormal splicing Alternative splicing occursby skipping exons.
Diverse proteins are made from
alternatively spliced mRNAs,which all are made
from a gene.
Other forms of diversity
are alternative start sites
of transcriptionand translation.
Many of these are
subject to regulation.
Abnormal splicing can
occur by some mutationsor polymorphisms in DNA
that inactivate normal
splice sites or activate
cryptic splice sites.
Some are associated withdiseases.
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Self-splicingTwo sequential
transesterification
reactions at a site
Self-splicing RNA
has to fold into a
proper structurewithout the help of
spliceosome.
Thus, most of the
intron sequences
are critical in
selfsplicing,
whereas
they are not critical
in spliceosome
splicing.
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Processing at 3' end and nuclear transport
3'-Cleavage and
polyadenylationPoly(A) polymerase does not
require a template.
After 3'-cleavage, RPase II
continues for some distance.
Poly(A)-binding proteins mark polyadenylation.Nuclear export receptor is loaded on mature mRNA.
RNA debris are degraded by nuclear exosome.
Nuclear transport
"export-ready nuclear pore
RNA complex
CPSFCstF
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Non-coding RNA synthesis and processing in nucleolus
In nucleolus, some ribonucleoprotein
complexes (RNAs + proteins) areassembled (e.g. ribosomal subunits,
U6 snRNP, telomerase, signal
recognition particle, etc.) and tRNAs
are processed.
snoRNA (encoded by introns) and snRNA
produced mostly by RPase II aremodified at Cajal bodies and GEMS.
In nucleolus, pre-rRNA is produced
by RPase I, modified, cleaved into 3
rRNAs, and complexed withribosomal proteins.
(5S rRNA is produced by RPase III
and not modified.)
Subnuclear structures
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Figure 6-47. The function
of the nucleolus in
ribosome and other
ribonucleoproteinsynthesis.
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Terms:CTD carboxyl terminal domain
SR proteins -serine/arginine rich proteins
hnRNPs -heterogeneous nuclear ribonuclear proteinsCstF -cleavage stimulation factor F
CPSF -cleavage and polyadenylation specificity factor
Additional subnuclear structures:1.Cajal bodies (named for scientist who first described them in 1906)
2.GEMS (Gemini of coiled bodies)
-resemble one another and are frequently paired in the nucleu
-may be sites where snRNAs and snoRNAs undergo their final
modifications and assembly with protein-also sites where the snRNPs are recycled and their RNAs
are reset after the rearrangements that occur during splicing
3. interchromatin granule clusters (also called speckles)
-stockpiles of fully mature snRNPs that are ready to be used ins licin of re mRNAs
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