RNA Metabolism

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RNA Metabolism

• DNA-dependent synthesis of RNA
• RNA processing
• RNA-dependent synthesis of RNA DNA

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RNA (Ribonucleic Acid)

• Transcription an enzyme system converts the
  genetic information in dsDNA into an RNA strand
  with a base sequence complementary to one of the
  DNA strand.
• messenger RNA (mRNA)
• transfer RNA (tRNA)
• ribosomal RNA (rRNA)

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RNA Is Synthesized by RNA Polymerase

• Transcription in E. coli
• encompasses 35 bp of DNA (revealed by
  footprinting, p.985)
• requires DNA template, NTP Mg2
• adds nucleotide units to the strands 3-OH end
  in 5 3 direction

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17 base pairs of DNA template are unwound
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Supercoiling of DNA brought about by
transcription Positive supercoils form ahead of
the transcription bubble, and negative
supercoils form behind.
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The coding strand for a particular gene may
be located in either strand of a given
chromosome. e.g., adenovirus genome (36,000
bp) Many of the mRNA are initialy synthesized as
a long transcript (25,000 nt), which is then
extensively processed to produce the separate
mRNA.
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Structure of E. coli RNA polymerase
Lacks 3 5 exonuclease activity error 10-4
to 10-5
holoenzyme
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RNA Synthesis Is Initiated at Promoters
Consensus sequence of typical E. coli
promoters recognized by RNA polymerase
holoenzyme containing s70
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RNA Polymerase Leaves Its Footprint on a
Promoter Footprinting a method that provides
information about the interaction between RNA
polymerase and promoters.
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Specific Sequences Signal Termination of RNA
Synthesis

• Not yet well understood in eukaryotes
• At least two signals in E. coli
  r(rho)-dependent and r-independent

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r-independent termination of transcription
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Eukaryotic Cells Have Three Kinds of Nuclear RNA
Polymerase

• RNA polymerase I rRNA
• RNA polymerase II mRNA etc.
• RNA polymerase III tRNA, 5S rRNA etc.

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Common Sequences in Promoters Recognized by
Eukaryotic RNA Polymerase II
Initiator sequence
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RNA Polymerase II Requires Many Other Protein
Factors for Its Activity
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Transcription at RNA Polymerase II Promoters

• assembly
• initiation
• elongation
• termination

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The Structure of TBP (gray) Bound to DNA (blue
and white)
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RNA Polymerase Can Be Selectively Inhibited

• actinomycin D prok/euk.
• rifampicin prok.
• a-amanitin euk. pol II etc.

Inserted into DNA between G/C
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A Complex of Actinomycin D and DNA
G
C
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RNA Metabolism

• DNA-dependent synthesis of RNA
• RNA processing
• RNA-dependent synthesis of RNA DNA

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Maturation of mRNA In a Eukaryotic Cell
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Phillip Sharp Richard Roberts, 1977 The genes
for polypeptides in eukaryotes are often
interrupted by noncoding sequences
(introns). i.e., split gene
e.g., chicken ovalbumin gene
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Chicken ovalbumin gene
Introns are removed by splicing
intron A-G exon 1-7
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Introns

• Group I guanosine 3OH as nucleophile
• Group II adenosine 2OH in intron as nucleophile
• Group III dependent on snRNPs, pronounced
  snurps (small nuclear ribonucleoproteins), not
  self-splicing
• Group IV need ATP and endonuclease

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RNA Catalyzes Splicing Thomas Cech et al., 1982
(p.994) protozoan Tetrahymena thermophila the
splicing mechanism of group I rRNA intron Sidney
Altman et al., 1983 (p.1004) E. coli M1 RNA (377
nt) of RNase P cut tRNA
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Transesterification reaction the first step in
the splicing of group I introns
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Splicing mechanism of group I introns
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Splicing mechanism of group II introns
lariat
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Splicing mechanism of group III introns in
eukaryotic mRNA primary transcripts
snRNAs (small nuclear RNAs)
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Assembly of spliceosomes snRNPs (snurps)
snRNA-protein complexes
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Splicing mechanism of group IV introns in yeast
tRNA
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Eukaryotic mRNA Undergo Additional Processing

• adding 5 cap
• adding poly(A) tail

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cap
7-methylguanosine is added to the 5 end of
almost all eukaryotic mRNAs in 5,5-triphosphate
linkage. Methyl groups (red) are sometimes
found at the 2 position of the first and second
nt. (not in yeast)
first second
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Generation of the 5 cap
adoMet S-adenosylmethionine
adoHcy S-adenosylhomocysteine
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Addition of the poly(A) tail to the primary RNA
transcript of eukaryotes
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Overview of the processing of a eukaryotic mRNA
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Multiple Products Are Derived from One Gene by
Differential RNA Processing
Alternative cleavage polyadenylation
Alternative splicing
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E.g., Alternative processing of the calcitonin
gene transcript in rats
(calcitonin-gene-related peptide)
calcium-regulating hormone
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rRNAs and tRNAs Also Undergo Processing
Processing of pre-rRNA in bacteria
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Processing of pre-rRNAs in vertebrates
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Processing of tRNAs in bacteria eukaryotes
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Some modified bases of tRNAs, produced in
post-trancriptional reactions
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Some Events in RNA Metabolism Are Catalyzed by
RNA Enzymes
Hammerhead ribozyme (only 41 nucleotides) requires
Mg2
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E.g., the self-splicing rRNA intron from
Tetrahymena
Internal guide sequence (boxed) pairs with splice
site at 5 end (red arrow) 3 end (blue arrow)
Intron (yellow) exon (green) catalytic core
(shaded)
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L-19 IVS (intervening sequences) has catalytic
activity in vitro, but quickly degraded in vivo.
L-19 IVS is generated by the autocatalytic
removal of 19 nt from 5 end of the spliced
intron.
(414 nt)
(395 nt)
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RNA enzymes L-19 IVS, from group I introns,
lengthens some RNA oligonucleotides at the
expense of others in a cycle of esterification
reaction
Oligo C paired with the same G-rich internal
guide sequences
L-19 IVS
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RNA Processing

• ...
• Cellular mRNA Are Degraded at Different Rates
• by ribonucleases usually in a 5 3
  direction
• occasionally in a 3 5 direction.
• In bacteria a hairpin structure in mRNA
  with r-independent terminator (p.986) confers
  stability.
• In eukaryotes the 3 poly(A) tail confers
  stability.
• A major pathway shortening the poly(A) tail
  gt decapping the 5 end gt degrading the RNA in the
  5 3 direction.

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Polynucleotide Phosphorylase Makes
Random RNA-like Polymers Marianne
Grunberg-Manago Severo Ochoa, 1955 (NMP)n
NDP (NMP)n1 Pi
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RNA Metabolism

• DNA-dependent synthesis of RNA
• RNA processing
• RNA-dependent synthesis of RNA DNA

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Extension of the central dogma to
include RNA-dependent synthesis of RNA and DNA
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Retroviral infection of a mammalian cell and
integration of the retrovirus into the host
chromosome
Reverse transcriptase
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Reverse Transcriptase Produces DNA from Viral
RNA Howard Temin David Baltimore,
1970 genetic information can flow backward
from RNA to DNA
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Structure and gene products of an integrated
retrovirus genome
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Retrovirus Cause Cancer and AIDS
Rous sarcoma virus genome
Peyton Rous RSV from chicken sarcoma,
1911 Harold Varmus Michael Bishop src oncogene
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Retrovirus Cause Cancer and AIDS
The genome of HIV, the virus that causes AIDS
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Fighting AIDS with Inhibitors of HIV Reverse
Transcriptase
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Many transposons, Retroviruses, and Introns May
Have a Common Evolutionary Origin
Eukaryotic transposons structurally similar to
retroviruses, but lacking env gene.
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Many transposons, Retroviruses, and Introns May
Have a Common Evolutionary Origin Introns that
move
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Many transposons, Retroviruses, and Introns May
Have a Common Evolutionary Origin Introns that
move
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Many transposons, Retroviruses, and Introns May
Have a Common Evolutionary Origin Introns that
move
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Telomerase Is a Specialized Reverse Transcriptase
The internal template RNA binds to and base-pairs
with the DNAs TG primer
Adding more T G
Reposition of the internal template RNA
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Telomerase Is a Specialized Reverse Transcriptase
How to protect ssDNA end?
Form T loops in telomeres (103 bp) of higher
eukaryotes including mammals
By specific binding proteins in telomeres (102
bp) of lower eukaryotes
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EM of a T loop of chromosome end from mouse
hepatocyte
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Some Viral RNAs Are Replicated by RNA-Dependent
RNA Polymerase

• Some E. coli RNA viruses, e.g., f2, MS2
• have RNA-dependent RNA polymerase (RNA replicase)
• which contains four subunits (210-kDa)
• one viral replicase for replication,
• three host proteins (elongation factors Tu
  and Ts, and
• 30S ribosome protein S1) for locating the
  3ends of the
• viral RNA

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RNA Synthesis Offers Important Clues to
Biochemical Evolution Carl Woese, Francis Crick
Leslie Orgel, 1960s theory RNA might serve as
both information carrier catalyst Thomas Cech
et al. Sidney Altman et al., 1980s proof
catalytic RNAs gtgt RNA world might have been
important in the transition from
prebiotic chemistry to life!
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Possible prebiotic synthesis of adenine from
ammonium cyanide
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RNA World Hypothesis Can a ribozyme replicate in
a template-dependent manner? The first step
making a ribozyme Reversible attack of a
guanosine on the 5 splice site in the removal of
the self-splicing group I intron (i.e., ribozyme
P1 region, p.1003)
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RNA World Hypothesis Can a ribozyme replicate in
a template-dependent manner? The ribozyme makes
template RNA capable of further RNA
polymerization reactions It can link oligo-RNAs
in a process equivalent to the reversal reaction
in (a)
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The ribozymes found in nature have a limited
repertoire of catalytic functions, but the
catalytic potential of RNA is far greater.
Rapid search for pools of random polymers of
RNAs with new catalytic functions is required!
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The search for RNAs with ATP-binding functions
by SELEX (systematic evolution of ligands by
exponential enrichment)
25 nt oligo in maximum 425 1015 random RNA
oligos
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ATP-binding RNA oligonucleotide isolated by SELEX