RNA Synthesis Gerald Wilson, Ph'D' gwils001umaryland'edu

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RNA SynthesisGerald Wilson, Ph.D.gwils001_at_umar
yland.edu
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Flow of Genetic Information The Central Dogma
of Molecular Biology
Alberts et al, 2002, p. 301
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What is RNA?

• RNA is a polymer composed of alternating units of
  ribonucleotides connected through a 3-5
  phosphodiester bond.

Nelson Cox, 2005, p. 277
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What is RNA (continued)?

• In contrast with DNA, ribonucleotides contain
• hydroxyl groups on the 2-carbon of the ribose
  sugar
• the base uracil in place of thymine

Alberts et al, 2002, p. 303
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RNA molecules fold into complex structures
Nelson Cox, 2005, pp. 289-290
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Types of RNA total
cellular RNA mass Ribosomal RNA
(rRNA) 85 -the RNA structural
component of the ribosome -in eukaryotes
there are 4 major forms 28S, 18S and 5.8S and
5S -in prokaryotes there are only 3 23S,
16S, and 5S S refers to a Svedberg Unit, which
is a measure of size based upon the
molecular sedimentation rate during
ultracentrifugation Messenger RNA (mRNA)
2 -the RNA that transfers genetic
information stored in DNA into a form
useable for protein synthesis Transfer RNA
(tRNA) 12 -assists in decoding the
information contained within mRNA during
translation by recruiting the correct amino acid
to the growing peptide chain Other forms
(snRNA, snoRNA) 1 -small nuclear RNAs
that participate in RNA processing
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Definitions and Conventions
Wilson, 2003
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General features of genes
Wilson, 2003
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General mechanism of RNA synthesis

• elongation by addition of ribonucleotides to the
  3-OH end
• 3-OH acts as a nucleophile, attacking the
  a-phosphate of the incoming ribonucleoside
  triphosphate and releasing pyrophosphate
• mechanism is the same as that used for elongation
  of a DNA strand (below)

Nelson Cox, 2005, p. 953
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General properties of DNA-dependent RNA
polymerases
(i) Polarity RNA polymerase reads the DNA
template in the 3?5 direction while
synthesizing RNA in the 5?3 direction
Wilson, 2003

• (ii) DNA template Either strand of a DNA double
  helix can serve as a template for RNA synthesis.

Nelson Cox, 2005, p. 998
(iii) Fidelity RNA polymerases do not possess
3?5 proofreading activities RNA
polymerase error rate 1 in 104 105 nt DNA
polymerase error rate 1 in 109 1010 nt
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(iv) Mechanical features of RNA synthesis
Nelson Cox, 2005, p. 996
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(v) Role of topoisomerases
Nelson Cox, 2005, p. 997
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E. coli RNA polymerase
36.5kD
151kD
11kD
155kD
36.5kD
70kD
Nelson Cox, 2000, p. 982
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E. coli RNA polymerase
Nelson Cox, 2005, p. 999
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Identification of protein-binding sites on DNA by
nuclease footprinting
Nelson Cox, 2005, p. 1002
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Nelson Cox, 2005, p. 1002
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Prokaryotic promoter elements
Nelson Cox, 2005, p. 999
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Transcriptional Initiation in E. coli
Nelson Cox, 2005, p. 1000
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Rho-independent transcriptional termination
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Nelson Cox, 2005, p. 1001
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Rho-dependent transcriptional termination

• RNA polymerase transcribes DNA.
• Rho attaches to its recognition site on RNA.
• Rho moves along RNA, following the polymerase
  RNA polymerase pauses at terminator and rho
  catches up.
• Rho unwinds the DNARNA hybrid in the
  transcription bubble.
• Termination RNA polymerase, rho, and RNA are
  released.

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Eukaryotic RNA Polymerases
Polymerase Products Polymerase I (Pol I)
rRNAs 28S, 18S and 5.8S Polymerase II (Pol
II) mRNA, some small RNAs Polymerase III
(Pol III) tRNAs and 5S, additional small
RNAs

• Unlike the E. coli RNA polymerase holoenzyme,
  each of these require a number of additional
  proteins called transcription factors in order
  to specifically bind to a promoter and initiate
  transcription.
• Eukaryotic promoters are composed of a variety of
  different cis sequence elements which recruit
  some of these trans-acting factors through
  DNA-protein interactions.
• Protein-protein interactions also occur and
  account for many of the multi-component complexes
  found at eukaryotic promoters.
• Pol I and III promoters utilize a small number of
  ubiquitous transcription factors while Pol II
  uses a large variety of specific ones.

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Initiation at RNA Pol I promoters

• One model
• Two identical subunits of the upstream binding
  factor bind to the upstream core element and the
  core promoter element.
• Proteinprotein interactions between UBF
  molecules force these two DNA sequences to come
  into close proximity.
• This enables subsequent binding of selectivity
  factor I, which consists of four subunits.
• Ultimately, this stabilized structure permits
  binding of other factors (not shown), and finally
  RNA pol I.

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Initiation at RNA pol III promoters
Positions of promoter elements in tRNA and 5S
rRNA genes.

• Initiation of transcription of a tRNA gene
• The TFIIIC transcription factor binds via
  recognition of the A and B sites
• This permits subsequent binding of the trimeric
  TFIIIB factor immediately upstream of the
  transcription start site.
• In response to TFIIIB binding, RNA polymerase III
  is recruited and initiates transcription.

In the case of 5S rRNA genes, the process is
similar, except that an additional factor,
TFIIIA, is required. TFIIIA binds the C box,
which permits subsequent binding of TFIIIB and
TFIIIC, then recruitment of RNA pol III.
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Initiation at RNA pol II promoters
Nelson Cox, 2005, p. 1004
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Conserved locations in complex eukaryotes for
regulatory promoter elements bound by ubiquitous
transcription factors
NB The core promoters of individual genes do not
require all of these elements. For example, many
promoters lack a TATA box, using instead the
functionally analogous initiator (INR)
element. Abbreviations BRE, TFIIB recognition
element DPE, downstream promoter element CTF,
CCAAT-binding transcription factor CBF,
CCAAT-box-binding factor TBP, TATA box-binding
protein TAF, TBP-associated factors.
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Structure of TBP bound to DNA
Nelson Cox, 2005, p. 1004
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Transcription Inhibitors

• actinomycin D, acridine
• -intercalate between successive GC base pairs
  in duplex DNA
• -inhibit transcriptional elongation in pro- and
  eukaryotes
• rifampicin
• -binds the b subunit of bacterial RNA polymerase
• -blocks promoter clearance (elongation)
• a-amanitin
• -produced by fungus Amanita phalloides (death
  cap mushroom)
• -potent inhibitor of RNA pol II and weak
  inhibitor of RNA pol III

Nelson Cox, 2005, p. 1006
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Complex of actinomycin D with DNA
Nelson Cox, 2005, p. 1006