Transcription: Synthesis of RNA

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Transcription Synthesis of RNA
J. Paul Simon
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Learning Objectives
Describe the two main chemical compositional
differences between DNA and RNA
Define the following terms as they apply to RNA
synthesis Template and coding strand Promoter
sequence Primary mRNA transcript Introns and
exons RNA splicing
Describe the function of the three major types of
RNA Messenger RNA Transfer RNA Ribosomal RNA
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Describe the sequence of events in transcription
with respect to the following Binding and
initiation Closed and open promoter Termination
Describe the mechanism of action of the
transcription inhibitors rifampicin and
actinomycin D.
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Structure of RNA
RNA is ribonucleic acid. Like DNA, RNA is a
polynucleotide chain in which the nucleotides are
linked by phosphodiester bonds.
Purine and pyrimidine bases A, U, G, C
Ribose sugar
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RNAs function as single strands. However,
these strands fold back upon themselves with the
potential for much greater structural diversity
than DNA.
Three-dimensional structure of phenylalanine tRNA
from yeast
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Major types of RNA
Messenger RNA (mRNA) encodes the amino acid
sequence of one or more polypeptides specified by
a gene or set of genes.
Transfer RNA (tRNA) reads the information
encoded in the mRNA and transfers the appropriate
amino acid to a growing polypeptide chain during
protein synthesis.
Ribosomal RNA (rRNA) these are constituents of
ribosomes, the intricate cellular machines where
proteins are synthesized.
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Transcription
An enzyme system converts genetic information in
a segment of double stranded DNA into an RNA
strand with a base sequence complimentary to one
of the DNA strands.
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The DNA strand that serves as template for RNA
synthesis is called the template strand.
The DNA strand complementary to the template, or
coding strand, is identical in base sequence to
the RNA transcribed from the gene with U in place
of T.
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Structure of the E. coli DNA-dependent RNA
polymerase
This is a large complex enzyme with five core
subunits and a sixth subunit, one of a group
designated s. The s subunit binds transiently to
the core and directs the enzyme to specific sites
on the DNA (promoter). The major type is s70.
The active site for RNA synthesis is thought to
be formed by the b and b subunits.
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Typical E. coli promoters recognized by RNA
polymerase with s70
The upstream promoter element (UP) occurs in the
promoters of certain highly expressed genes.
These
elements strongly stimulate transcription at the
promoters that contain them. (The promoter with
the UP is for a highly expressed ribosomal RNA).
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Initiation of transcription by E. coli RNA
polymerase
Initiation requires several steps divided into
two phases binding and initiation. In the
binding phase, RNA polymerase binds to the DNA at
the promoter and forms a closed complex the
promoter is stably bound but not open (closed
promoter). The region of DNA from -10 to 2 or 3
is then unwound to form an open complex (open
promoter). Once the first 8 or 9 nucleotides of
a new RNA are synthesized, the s subunit is
released and the polymerase leaves the promoter
region and becomes committed to elongation of the
RNA (promoter clearance).
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Mechanism of action of rifampicin
Rifampicin inhibits bacterial RNA synthesis by
binding to the b subunit of bacterial RNA
polymerase, preventing the promoter clearance
step of transcription. As such, it inhibits
initiation of transcription.
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Rho-independent termination in E. coli
RNA polymerase pauses at a variety of sequences,
some of which are terminators. A terminator
sequence is a region where the RNA transcript has
self-complimentary sequences permitting the
formation of a hairpin structure. It also has a
short string of adenylates (A) that are
transcribed into uridylates at the 3 end of the
RNA. Formation of the hairpin structure
facilitates dissociation of the RNA transcript.
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Rho-dependent termination in E. coli
The rho-dependent terminator sequences lack the
repeated adenylates in the template strand, but
sometimes have a sequence that can form a
hairpin. The rho protein loads onto the RNA and
migrates in the 5 to 3 direction until it
reaches a polymerase complex paused at a
termination sequence. Here it contributes to the
release of the transcript. ATP is hydrolyzed by
rho protein during the termination process.
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Eukaryotic RNA polymerases
RNA polymerase I is responsible for only one
type of RNA, a transcript called preribosomal
RNA. This contains the precursors for the 18S,
5.8S, and 28S rRNAs. RNA polymerase II functions
to synthesize mRNAs and some specialized
RNAs. RNA polymerase III makes tRNAs, the 5S
rRNA, and some small specialized RNAs.
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Proteins required for transcription at eukaryotic
promoters
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Transcription at eukaryotic RNA polymerase II
promoters
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Mechanism of action of actinomycin D
The elongation of RNA strands in both bacteria
and eukaryotes is inhibited by actinomycin D.
The planar portion of this molecule intercalates
into the double helical DNA between successive GC
base pairs, deforming the DNA. This prevents the
movement of the polymerase along the template.
The DNA backbone is blue, bases are white. The
intercalated part of actinomycin D is orange. The
two cyclic peptide structures (red) bind to the
minor groove of DNA. The DNA is bent as a result
of binding of actinomycin D.
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Maturation of mRNA in a eukaryotic cell
The 5 cap is added before synthesis is complete
Splicing can occur before or after polyadenylation
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5 Cap of mRNA
7-Methylguanosine is joined to the 5 end of
almost all eukaryotic mRNAs in an unusual
5,5-triphosphate linkage. Methyl groups (red)
are often found at the 2 positions in the first
and second nucleotides.
The 5 cap binds to a specific protein and may
participate in the binding of the mRNA to the
ribosome. It is likely that the 5 cap and the
polyA tail and their associated proteins help
protect the mRNA from enzymatic degradation.
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RNA Processing in Eukaryotes
Exon A segment of a eukaryotic gene that encodes
a portion of the final product of the gene a
portion that remains after posttranscriptional
processing and is translated into protein or
incorporated into the structure of an
RNA. Intron a sequence of nucleotides in a gene
that is transcribed but excised before
translation. RNA splicing Removal of introns
from a primary RNA transcript, and subsequent
joining of exons. Spliceosome a complex of RNAs
and protein involved in the splicing of mRNAs in
eukaryotic cells.
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Demonstration of noncoding sequences in the
chicken ovalbumin gene by DNA-RNA hybridization.
Exons are numbered. Introns are designated with
letters.
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Splicing mechanism of Group I introns
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Splicing mechanism of Group II introns
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Group I and group II introns are self-splicing.
No protein enzymes are involved. RNAs can have
catalytic functions.
Most of the eukaryotic nuclear mRNA primary
transcripts require the action of specialized
RNA-protein complexes called small nuclear
ribonuclearproteins (snRNPs or snurps). Each
snRNP contains one of a class of RNAs called
small nuclear RNAs. These are 100-200
nucleotides long and are designated as U1, U2,
U3, U4, U5, and U6. These RNAs and about 50
proteins make up the spliceosome.
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Splicing mechanism in mRNA primary transcripts
The U1 snRNA has a sequence near its 5 end that
is complimentary to the splice site at the 5 end
of the intron. Base pairing of U1 to this region
helps define the 5 splice site. U2 is paired to
the intron at a position encompassing the
adenosine residue that becomes the nucleophile
during the splicing reaction. Base pairing of U2
causes a bulge in the mRNA that displaces and
helps to activate the adenosine.
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Spliceosome assembly
The adenosine 2 hydroxyl is the nucleophile to
form the lariat structure. (Similar to Group II
introns)
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Addition of the poly(A) tail to the primary mRNA
transcript of eukaryotes
RNA polymerase synthesizes RNA beyond the
cleavage signal sequences, including the highly
conserved AAUAAA. (a) The cleavage signal
sequence is bound by an enzyme complex that
includes an endonuclease, a polyadenylate
polymerase, and several other multi subunit
proteins involved in sequence recognition, and
regulation of the length of the poly(A) tail. (b)
The RNA is cleaved by the endonuclease at a point
10 to 30 nucleotides downstream of the AAUAAA
sequence. (c) The polyadenylate polymerase
synthesizes a poly(A) tail 80 to 250 nucleotides
in length, beginning at the cleavage site.
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Overview of the processing of a eukaryotic mRNA
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Two mechanisms for the alternative processing of
complex transcripts in eukaryotes in both
mechanisms different mature RNAs are produced
from the same primary transcript.
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Processing of transfer RNA