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

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... example, many promoters lack a TATA box, using instead the functionally ... CCAAT-box-binding factor; TBP, TATA box-binding protein; TAF, TBP-associated factors. ... – PowerPoint PPT presentation

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


1
RNA SynthesisGerald Wilson, Ph.D.gwils001_at_umar
yland.edu
2
Flow of Genetic Information The Central Dogma
of Molecular Biology
Alberts et al, 2002, p. 301
3
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
4
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
5
RNA molecules fold into complex structures
Nelson Cox, 2005, pp. 289-290
6
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
7
Definitions and Conventions
Wilson, 2003
8
General features of genes
Wilson, 2003
9
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
10
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
11
(iv) Mechanical features of RNA synthesis
Nelson Cox, 2005, p. 996
12
(v) Role of topoisomerases
Nelson Cox, 2005, p. 997
13
E. coli RNA polymerase
36.5kD
151kD
11kD
155kD
36.5kD
70kD
Nelson Cox, 2000, p. 982
14
E. coli RNA polymerase
Nelson Cox, 2005, p. 999
15
Identification of protein-binding sites on DNA by
nuclease footprinting
Nelson Cox, 2005, p. 1002
16
Nelson Cox, 2005, p. 1002
17
Prokaryotic promoter elements
Nelson Cox, 2005, p. 999
18
Transcriptional Initiation in E. coli
Nelson Cox, 2005, p. 1000
19
Rho-independent transcriptional termination
5
Nelson Cox, 2005, p. 1001
20
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.

21
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.

22
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.

23
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.
24
Initiation at RNA pol II promoters
Nelson Cox, 2005, p. 1004
25
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.
26
Structure of TBP bound to DNA
Nelson Cox, 2005, p. 1004
27
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
28
Complex of actinomycin D with DNA
Nelson Cox, 2005, p. 1006
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