DNARNAPTN

1

• DNA RNA PTN
• Transcription process of making RNA from a DNA
  template
• (DNA dependent RNA synthesis). RNA Polymerase
• Types of RNA
• Informational Used to make polypeptides
• A. mRNA
• 1. Prokaryotic. mRNA made off DNA used
  directly in TLN
• 2. Eukaryotic. Primary transcript made then
  PROCESSED to give mature mRNA (RNA processing)
• IIgt Functional RNA do not encode polypeptides
  (these are gene PRODUCTS )
• Include tRNAs (transporter RNA TLN machinery),
  rRNA (ribosomal RNA, with ptns make up ribosomes,
  TLN site), snRNA (splicing RNA, make RNPs to
  serve as platforms for splicing rxns), scRNAs
  (small cytoplasmic RNAs, direct traffic in
  cytosol)
• DNA/RNA Function based on 2 features
  Complementarity laws and DNA or RNA specific
  binding ptns (recognize DNA or RNA sequence
  either DS or SS)

2

• Properties RNA
• RNA usually single stranded (has high degree of
  folding however)
• RNA has RIBOSE sugar (text p 300) -OH at 2 (-H
  in deoxy)
• RNA has AUGC (U replaces T found in DNA) AU
  pairs however
• DNA is nuclear in Eukaryotes PTN is made
  cytoplasm thus RNA likely intermediate.

Early Expts using 3H Uridine pulse chase expts
autoradiography
Pulse/Chase Allows one to track a synthetic
event over time RNA appears to be made in
Nucleus and transported to cytosol -Similar
expts. With phage showed that RNA turns over (has
a lifetime (also RNA sequence matches DNA
sequence)
3
Conclusion from foregoing data

• EM of TXN rRNA synthesis, note
• Christmas tree appearance
• Many RNA Pol. Molecules
• Short RNA at start of gene (5 end)
• Longer RNA at end of gene

4
Txn is usually asymmetrical 1 strand or other
is template for RNA (defines template vs.
nontemplate or coding vs non coding strands).

• NOTE
• Complementarity
• Asymmetric Txn
• No T in mRNA product (A calls for U)
• Polarity of synthesis.

5
Txn of two genes How txn organization may work
inside cells
Above illustrates polarity of the process off
template (coding) strand (always 5 to 3, thus,
the DNA template strand is oriented 3 to 5
6
RNA Polymerase (Rpol) in prokaryotic organisms
single Rpol (E. coli shown). Organization is
important to process of Initiation, elongation
and termination. Note that even in prokaryotes
Rpol is large and complex enzyme with many
co-factors and subunits. NEXT Initiation,
Elongation and Terminationgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt
Initiation factor allows initiation at proper
sites, then dissociates.
7
Initiation DNA regions that specify txn start
sites (also regulate txn) called promoters.
These are special sequences as shown below
(bacterial)

• Key points
• Promoter is 5 of gene (recall txn takes place on
  COMPLEMENTARY strand)
• Shaded areas consensus or conserved sequences
• Plus 1 site is first base of mRNA
• Consensus seq. shown for all bacterial promoters

8
Two regions of promoter homology -35 and 10
These are sites of Rpol CONTACT in the promoter
region. Rpol actually unwinds DNA at or near
this site
Initiation starts with Rpol linear search of the
DNA to locate promoter
Rpol linear search leads to closed complex
binding at 10/-35 region promoter
Rpol forms open complex melt out of region near
10 Txn intitates from open complex and once
initiated, sigma can dissociate
All of the above require SIGMA factor
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ELONGATION NTP (NMP)n (NMP)n1 PP
(inorganic Phosphate release provides energy for
forward synthesis).
DNA, Mg, RPol
Note Txn bubble maintained as synthesis
proceeds
Recall must have SS DNA to read sequence
information!!
10
TERMINATION OF TXN
Rpol recognizes signals for termination of RNA
chains. This releases the enzyme and RNA
product Two mechanisms in bacteria DIRECT
Terminator seq. (ca. 40 bp) GC rich seq. followed
by a run of 6 or more As (template DNA strand
thus, U rich on RNA). Hairpin loop forms with
the RNA structure this signals the release of
the Rpol and termination of txn.
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TERMINATION OF TXN
Rpol.
Second mechanism RHO dependent termination A
ptn. Cofactor call rho is added to Rpol which
facilitates termination signal recognition.
There are no hairpin features in RNA as seen in
DIRECT termination
ribosome
Rhorut

• Second mechanism
• Rho Hexamer uses ATP to drive termination.
• Rho binds to a seq. in RNA called rut
• Rho/rut pulls RNA off Rpol using ATP energy
• Rut sites located 5 of pause sites

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Eukaryotic RNA
Rpol 3 in eukaryotes Rpol I rRNA Rpol II
mRNA (mRNAs always monocistronic) Rpol III tRNAs
and snRNAs
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• RNA Processing
• Txn product called primary transcript
• matured into mRNA
• Transported to Cytosplasm
• Tln

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• Details of processing
• 5 Cap m7Gppp (guanyltransferase)
• 3 PolyA (150-200)

5Cap
AAUAAA Signals Cleavage of nascent transcript
ca. 20 nt downstream
Poly A polymerase adds 3 A
Splicing
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Split Genes
SV40 expts transcripts did not always
correspond to linear DNA sequencerecombinant DNA
technology confirmed that internal sequences were
missing in the mRNA (from the primary
transcript). Found with mRNA, tRNA and rRNA as
well Ovalbumin Gene (egg white ptn) EM diagram
of GENE with mature mRNA
Intron
Exon
Intron
Introns Intervening sequences Exons Coding
sequencesA series of discrete splicing steps cut
and reseal the transcript to make mRNA
Exon
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Entire sequence of events for RNA processing and
splicing. ALTERNATIVE SPLICING Produces
different mRNAs and proteins from the SAME
primary transcript. Lots of genetic flexibility
and possibilities.
17
Alternative splicing with tropomyosin Different
cell types have alt. Spliced forms of the gene
(light green introns). -Ptns clearly related
but function differently in each cell lineage.
Poly A signals
Exons
Cell Types/tissues
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Mechanism of Splicing Sequence homologies are
KEY!
At the 3 splice site AGOther consensus
elements further upstream help define where these
splice joints exist.
Small nuclear RNAs (snRNAs) act as guides in the
splicing process Forms the splicosome
Most begin with GU end with AG at the flanks
19
2 transesterification reactions releasing an
excised intron as a lariat structure.
20
Self splicing/Splicosome mechanistic details
Self-splicing (ribozymes) in gps. I and II.Group
I Introns T-4, Tetrahymena, mitochondria.
Group II some tRNAs and chroloplasts/mitochondri
al transcripts. Splicosome-catalyzed mRNA
GTP cofactor
Transesterification reactions
Spliced out intron is linear
Spliced out introns branched
21
Translation
mRNA directs synthesis of a polypeptide Action
happens in cytoplasm and on polysomes (ribosomes
mRNA)
22
How was tln figured out? Early fractionation of
macromolecules based upon the SIZE and SHAPE of
the structures in velocity sedimentation
gradients (or SUCROSE gradient centrifugation).
Large stuff goes to bottom
S Svedbergs measurement of size of the
macromolecule
Resolving power of sucrose ID RNAs involved.
23
Genetic Code how bases are read in TLN process.
Each word in the code is based upon a stretch
of 3 basesthis is a codon (or triplet). The
genetic code is NON-OVERLAPPINGearly on this was
thought to be so. WHY? Because a single base
mutation would give only a single AA change with
this modelwith the overlapping code, point
mutations would alter three amino acids
Note Shifted reading frames are possible! Cell
could utilize gt1 reading frame to double or
triple coding potential.increasing genetic
capacity at each locus.
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Number of letters in the genetic code with 20
naturally occurring amino acids 43 64 (more
than enough to drive the 20 thus there are some
extra words. SUPPRESSOR MUTATIONS (counteract
another mutation or suppress the mutation) 1st
demonstration of triplets. T4 rII system
proflavin mutants (delete or insert 1
base) Example of Insertion ATCTTA ATCGTTA
to give ATCGTTAExample of Deletion ATCTTA
AT_TTA to give ATTTA
Treat rIIx mutants with proflavin again, plate on
E.coli K a very few plaques appeared to be wt
revertants these behaved as wt rII phage
however, when additioal genetic crosses were done
with the T4 system, it was shown that a 2nd
mutation had been created that suppressed the
first one. Suggested that the reading frame was
shifted by an insertion (rIIx) and then shifted
back by a deletion (or vice versa?)
rII mutant x cannot plate on K
NOT like WT!!
The proflavin induced mutants were in fact
FRAMESHIFT MUTATIONS
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Frameshifts shift the reading frame by
insertion/deletion of a single base. ATA ATC TTT
TTA insertion of a G after C now gives ATA ATC
GTT TTT A and ORF (open reading frame) is
shifted Another example THE FAT CAT ATE THE BIG
RAT delete the C in CAT THE FAT ATA TET HEB IGR
AT Non-sense downstream of the deletion In
the rII system of suppressors, here is what
happened WT CAU CAU CAU CAU CAU Insert an A
in 2nd codon CAU ACA UCA UCA UCA U
Shift in the reading frame nonsense mRNA
results and defective protein Suppressor of above
possible with a deletion mutation CAU ACA UCU
CAU CAU Restores the reading frame but still
not quite right CAU CAU CAU CAU CAU WT sequence
compared in green, 2 amino acids changed
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FROM SUPPRESSOR MUTATIONS.If the original FCO is
defined as plus and the suppressor as minus
it was shown that a could suppress a but
could never suppress another . BUT three
pluses always gave back WT as did three
minuses! THE GENETIC CODE APPEARS TO BE A TRIPLET
OF 3 NUCLEOTIDES! PROOF of a triplet code from
the sequence level, 3 deletions in a single gene
will restore the reading frame (as will 3 single
base insertions) CAU CAU CAU CAU CAU CAU CAU
delete orange bases gives CAU ACA UAU CAU CAU
CAU WT MUT MUT WT WT
WT The findings were confirmed at the amino acid
sequence level. Also the code was shown to be
degenerate. Most amino acids have more than one
defined codon (triplet). Thus, some are
specified by gt1 codon.