Title: RNA Synthesis Gerald Wilson, Ph'D' gwils001umaryland'edu
1RNA SynthesisGerald Wilson, Ph.D.gwils001_at_umar
yland.edu
2Flow of Genetic Information The Central Dogma
of Molecular Biology
Alberts et al, 2002, p. 301
3What is RNA?
- RNA is a polymer composed of alternating units of
ribonucleotides connected through a 3-5
phosphodiester bond.
Nelson Cox, 2005, p. 277
4What 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
5RNA molecules fold into complex structures
Nelson Cox, 2005, pp. 289-290
6Types 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
7Definitions and Conventions
Wilson, 2003
8General features of genes
Wilson, 2003
9General 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
10General 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
13E. coli RNA polymerase
36.5kD
151kD
11kD
155kD
36.5kD
70kD
Nelson Cox, 2000, p. 982
14E. coli RNA polymerase
Nelson Cox, 2005, p. 999
15Identification of protein-binding sites on DNA by
nuclease footprinting
Nelson Cox, 2005, p. 1002
16Nelson Cox, 2005, p. 1002
17Prokaryotic promoter elements
Nelson Cox, 2005, p. 999
18Transcriptional Initiation in E. coli
Nelson Cox, 2005, p. 1000
19Rho-independent transcriptional termination
5
Nelson Cox, 2005, p. 1001
20Rho-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.
21Eukaryotic 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.
22Initiation 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.
23Initiation 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.
24Initiation at RNA pol II promoters
Nelson Cox, 2005, p. 1004
25Conserved 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.
26Structure of TBP bound to DNA
Nelson Cox, 2005, p. 1004
27Transcription 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
28Complex of actinomycin D with DNA
Nelson Cox, 2005, p. 1006