5 11transcription-2013

Transfer of genetic information:
RNA structure and transcription
Prof. Dalė Vieželienė

Department of biochemistry
Room No. 229
e-mail: [email protected]
Central Dogma of Molecular Genetics
DNA → 1 → DNA → 2 → RNA → 3 → Protein
1. Replication – process by
which a copy of DNA is
1. Replication – process by which a copy of DNA is made (upon cell division).
made (upon cell division).
2. Transcription – process by which information is transcribed into RNA (same information–different
2. Transcription processchemical).
by which information is
3. Translation – process by which genetic information is converted to amino acid sequence of proteins.
transcribed into RNA
(same information
different chemical).
3. Translation – process by
which genetic information
is converted to amino acid
sequence of proteins.
RNA specific structure and
functions
 Bases are A,G, C and U – note substitution of U for T
 Sugar is ribose
Only single stranded.
Types of RNA:
•Ribosomal RNA (rRNA)- associated with proteins to form the ribosomes needed
for protein synthesis. rRNA represents 80% of total cellular RNA. There are three
main kinds of rRNA: 23S, 16S and 5S (the S stands for sedimentation coefficient).
•Messenger RNA (mRNA) - carries genetic information used for protein synthesis.
mRNA represents about 5% of the total RNA in a cell at any given time
•Transfer RNA (tRNA) - carries and delivers amino acids of growing chain of
proteins during protein synthesis. tRNA represents 15% of total RNA. There is at
least one tRNA for each of the 20 essential amino acids and duplicate tRNAs for
some
•Small nuclear RNAs (snRNA) are integral part of small nuclear
ribonucleoproteins in eukaryotic cells, and are involved in splicing reactions.
snRNAs are 100 to 200 nucleotides long
Levels of RNA structure
 Primary Structure of RNA - linear and single-
stranded polynucleotide chain.
 Secondary Structure of RNA. The single strand
can fold to assume a right-handed helical
conformation. Double-helical stem-loop
regions in RNA often form “hairpins”:
stem – base-paired nucleotides;
loop – noncomplementary nucleotides
 Tertiary Structure
Levels of RNA structure
Secondary (cloverleaf) and Secondary Structure of 5S
tertiary (L-shaped ) rRNA
structure of tRNA
Introduction to DNA-Dependent
Synthesis of RNA
• Synthesis of an RNA molecule: copying one strand of a
template sequence using base pairing between
nucleotides of the template (DNA) and the nucleotides
that are being incorporated into the transcript.
• Synthesis proceeds in the 5`3`direction.
• Transcription differs from replication in that it does not
require a primer and involves only limited segments of a
DNA molecule. Within transcribed segments only one
DNA strand serves as a template.
Transcription initiates both in prokaryotes and
eukaryotes from many more sites than replication.
There are many more molecules of RNA polymerase

per cell than DNA polymerase.
RNA polymerase proceeds at a rate much lower than

DNA polymerase (approx. 50-90 bases/sec for RNA
versus 1000 bases/sec for DNA).
Finally, RNA polymerase lacks 3`5`exonuclease

activity, and the error rate for transcription is higher
than that for DNA replication – one error for every 104
to 105 ribonucleotides. As many copies of an RNA are
produced and all RNAs are eventually degraded and
replaced, a mistake in an RNA molecule is of less
consequence to the cell than a mistake in the
permanent information stored in DNA.
Stages of transcription
There are 3 stages of transcription:
•initiation,
•elongation
•termination.
A promoter
•A promoter site is a sequence of bases about 10 to 35 bases before the
actual code for the mRNA.
•The promoter sites tell the polymerase where to attach, and they are
always available for binding.
TATA or “Pribnow” box (-10 bp upstream from transcription start site, rich in A-T)
-35 region (-35 bp upstream) from start site: TTGACA.
The position of transcription initiation differs slightly among various promoters, but
usually is 5-8 bp downstream from the invariant TATA box
Scheme of transcription
RNA polymerase
All RNA’s are made from DNA templates by RNA polymerase. NTP are added
in the 5'→3' direction:
RNA + NTP →→ RNA (n+1) + PPi
RNA polymerase needs three things to work:
1) double stranded DNA as a template is preferred, but single strand DNA can
also be used.
2) activated precursors in the form of ATP, GTP, CTP and UTP.
3) magnesium (Mg2+) ions. Mg2+ is probably around the activated nucleotides,

and is stabilizing the energetic tri-phosphate region of the nucleotides.
• No primer is needed to start synthesizing RNA.

• RNA polymerase has no exonuclease activity.
Initiation and
elongation steps of
transcription by
bacterial RNA
polymerase
Binding
• The RNA polymerase
holoenzyme binds relatively
weakly to the promoter DNA to
form a “closed complex”, in
which the promoter DNA is not
unwound
• The holoenzyme forms a more
tightly bound “open complex”,
characterized by a local opening
of about 17 bp of the DNA double
helix. Since the consensus TATA
box is A-T rich, its base pairs are
more easily disrupted during
opening.
Initiation
• The unwound DNA binds the initiating purine nucleoside
triphosphate, and RNA polymerase then catalyzes
formation of the first phosphodiester bond
• The enzyme translocates to the next position and continues
synthesis.
Elongation
• Once the first 8 nucleotides of a new RNA are synthesized,

the sigma subunit is released and the polymerase leaves the
promoter and enters the elongation phase
• Once the RNA polymerase has cleared the promoter region,
other RNA polymerase molecules can bind and initiate at the
promoter so that the gene can be transcribed by many
polymerases at the same time
Termination of
transcription
Transcription in Eukaryotic Cells
 Chromatin containing the promoter sequence must be
made accessible to the transcription machinery.
 Transcription factors distinct from RNA polymerase must
bind to DNA sequences in the promoter region for a gene to
be active. This contrasts with the action of bacterial sigma
factors, which do not bind DNA without first binding to the
RNA polymerase core enzyme.
 Enhancers bind other protein factors to stimulate
transcription.
• Few if any natural promoters contains all elements.

• Only 20-30% of promoters contain a TATA element.
• Human: ~32% of protein coding genes have TATA out of 1031 genes (Suzuki Y
et al Genome Res, 2001)
• 48% CpG island.
Eukaryotic RNA Polymerases
In eukaryotic cells there are four distinct RNA
polymerases. Each polymerase is responsible for the
synthesis of different type of RNA:
 RNA polymerase I – is concentrated in nucleolus and

transcribes 5.8S, 18S and 28S rRNAs.
 RNA polymerase II – synthesizes mRNA and some
snRNAs.
 RNA polymerase III –synthesizes tRNA, 5S rRNA and
snRNAs.
 Mitochondrial RNA polymerase – is responsible for
synthesis of mitochondrial mRNA, tRNA and rRNA
species.
Transcription at RNA Pol II Promoters
The core promoter binds to the general transcription factors and Pol
II and is the site for assembly of the preinitiation complex (PIC).
Core promoter gives low basal transcription in vitro but is generally
inactive in vivo.
RNA Processing
RNA processing is to generate a mature mRNA
(for protein genes) or a functional tRNA or
rRNA from the primary transcript
The reactions of RNA processing can include:
 removal of extra nucleotides,
 covalent base modification,
 addition of nucleotides,
 separation of different RNA sequences by specific nucleases.
Processing reactions can occur either co-transcriptionally (while the RNA
is still being transcribed) or posttranscriptionally (after the transcript is
released by RNA polymerase).
In eukaryotes, RNAs are exported from the nucleus
Processing of mRNA
Processing of pre-mRNA involves the

following steps:
•Capping - add 7-methylguanylate (m7G) to
the 5' end.
•Polyadenilation - add a poly-A tail to the 3'
end.
•Splicing - remove introns and join exons.
1. Capping
Capping occurs shortly after transcription
begins
2. Polyadenilation of mRNA
A stretch of adenylate residues are added to the 3' end. The poly-A
tail contains ~ 250 A residues in mammals, and ~ 100 in yeasts
The major signal for the 3' cleavage is the sequence AAUAAA. Cleavage occurs at 10-
35 nucleotides downstream from the specific sequence. A second signal is located
about 50 nucleotides downstream from the cleavage site. This signal is a GU-rich or
U-rich region.
3 - Splicing
RNA splicing is a process that removes introns and joins exons in a

primary transcript.
Splicing
Most introns start from the
sequence GU and end with
the sequence AG (in the 5' to
3' direction).
Another important sequence
is called the branch site
located 20 - 50 bases
upstream of the end of
introne. The consensus
sequence of the branch site is
"CU(A/G)A(C/U)", where A
is conserved in all genes.
Alternative splicing
An intron usually contains a clear signal for splicing In some
cases, a splicing signal may be masked by a regulatory protein,
resulting in alternative splicing.
Structure of mature mRNA
• Pre-tRNA requires extensive
Processing of processing to become a
functional tRNA.
tRNA • Four types of modifications
are involved:
• Removing an extra segment (~
16 nucleotides) at the 5' end by
RNase P.
• Removing an intron (~ 14
nucleotides) in the anticodon
loop by splicing.
• Replacing two U residues at the
3'end by CCA, which is found
in all mature tRNAs.
• Modifying some residues to
characteristic bases, e.g.,
inosine, dihydrouridine and
pseudouridine. Main
modificatio is methylation
Processing of rRNAs
rRNAs of both prokaryotic and eukaryotic cells are
made from longer precursors called preribosomal
RNAs (pre-RNA).
Transcription: Main points
 Information in DNA rewritten to form single stranded m-RNA

 Only one chain of DNA used in this process (ie only one chain
contains the genetic information and serves as a template).
 RNA Polymerase - catalyzes transcription
 Initiation of Transcription begins at "Promoter Sites" -
specific sequences recognized by RNA Polymerase
 Sequence of nucleosides in DNA is not contiguous for coding
for a protein.
o 1. Introns - intervening sequences
o 2. Exons - coding sequences.
o 3. Processing of RNA deletes intron sequences
and joins together the exon sequences.

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Transfer of genetic information:

RNA structure and transcription

Prof. Dalė Vieželienė

There are many more molecules of RNA polymerase

RNA polymerase proceeds at a rate much lower than

Finally, RNA polymerase lacks 3`5`exonuclease

There are 3 stages of transcription:

RNA polymerase needs three things to work:

2) activated precursors in the form of ATP, GTP, CTP and UTP.

3) magnesium (Mg2+) ions. Mg2+ is probably around the activated nucleotides,

• No primer is needed to start synthesizing RNA.

• Once the first 8 nucleotides of a new RNA are synthesized,

• Few if any natural promoters contains all elements.

 RNA polymerase I – is concentrated in nucleolus and

Processing of pre-mRNA involves the

RNA splicing is a process that removes introns and joins exons in a

 Information in DNA rewritten to form single stranded m-RNA

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