RNA and Transcription
(gene expression)
How is the information in DNA utilized?
The Central Dogma
Replication
DNA
RNA
Transcription
Protein
Translation
directional flow of information
RNA is a complex molecule that carries
codes, harbors enzymatic activity and
performs a structural function
Secondary structure of large subunit ribosomal RNA
Producing RNA
involves
chromatin
dynamics
DNA methylation
Methyl groups added to
certain DNA bases repress
gene activity.
Histone modification
A combination of different
molecules can attach to the
tails of proteins called
histones. These alter the
activity of the DNA wrapped
around them.
Compact
chromatin
DNA accessible to gene
expressing proteins
Goals and timing of DNA and RNA synthesis are very different
RNA synthesis is very dynamically regulated
Making many copies is a way to amplify the information present in DNA
RNA molecules
important intermediates in flow of
genetic information
Transfer RNA
Ribosomal RNA
Messenger RNA
Uracil vs. Thymine
Methyl
group
RNA strands use uracil, while DNA strands
use thymine
Uracil is a demethylated thymine
RNA
Ribonucleic acid
A molecule also made up
of nucleotides linked
together by covalent
bonds
DNA is transcribed into
RNA using the SAME
RULES of nucleotide
base pairing used by
DNA, except that Uracil
is used
H!
H!
H!
An RNA Nucleotide
Ribose, a monosaccharide
A phosphate group (PO4)
A nitrogenous base (either adenine,
guanine, cytosine, or uracil)
RIBONUCLEOTIDES,
NOT DEOXYRIBO
NUCLEOTIDES
RNA Transcription
Definition: the synthesis of RNA, using DNA
as a template
Occurs during interphase within the nucleus
Enzymes cause the DNA double-helix to uncoil
and straighten out
The weak hydrogen bonds between the base
pairs break apart (the molecule unzips )
One of the two strands of nucleotides is used as
the DNA coding strand; the nitrogenous
bases of this strand bond with new RNA bases
within the nucleus to form a strand of RNA
RNA Transcription (continued)
Cytosine and guanine bond together; adenine
and uracil bond together
Once the RNA is synthesized, the two strands
of DNA re-bond with one another; the DNA
returns to its coiled, double-helix shape
The RNA strand is modified into messenger
RNA (mRNA)
mRNA is transported out of the nucleus and
into the cytoplasm to be used for protein
synthesis
RNA polymerase (pol II)
RNA polymerase II
transcription-initiation complex
Initiation by Pol II requires general
transcription factors, which position Pol II at
initiation sites and are required for
transcription of most genes transcribed by this
polymerase
General transcription factors are multimeric
Proteins comprising the Pol II transcriptioninitiation complex assemble in a specific order
in vitro
Pre-initiation complex (PIC)
DNA
RNAP II: RNA polymerase II (RNApol II)
CTD: carboxy-terminal domain of RNAP II
TFIID 11: TBP+10 TAFs (TBP associated factors)
TFIIB 1: single peptide binds after TBP
TFIIF 4: stablilizes the complex TBP-TFIIB-RNAP II, necessary for TFIIE entry
TFIIE 4: helps TFIIH entry
TFIIH 9: has the catalytic activity (helicase and kinas cdk7-cyclinH)
* Numbers refer to the number of proteins in complex (subunits)
Control of transcription
Transcription start site usually a TATA
box (not always)
TBP (TATA-binding protein) binds,
changing DNA structure
Recruits transcription factor II proteins
(TFIIA, B, ) then RNA Pol II
Collectively known as the pre-initiation
complex or the transcription initiation
complex
Stepwise assembly of Pol II transcriptioninitiation complex in vitro!
CTD
RNA synthesized
Release of
transcription factors
The basic
mechanism of
transcribing DNA
into RNA!
Processing of Transcripts
Capping of 5 end of RNA
Poly-adenylate 3 end (add Poly A tail):
Poly A polymerase adds 100 s of
adenosines (A s) to end of transcript;
length of poly A tail influences half-life of
RNA (degradation rate)
Splice out intervening sequences
(introns), leaving expressed sequences
(exons)
Eukaryotic primary RNA transcripts are
modified by addition of a 5 methylated cap"
Unique linkage!
recognized by special factors!
not susceptible to nucleases!
Poly A tail
RNA transcripts contain AAUAAA site at
the 3 end
RNA is cleaved 10-36 nucleotides
downstream of the AAUAA site
Poly(A) polymerase adds a tail of 100-200
adenosines at the 3 end of the cleaved
RNA strand, just passed the AAUAA site
Poly A tail used to protect mRNA from
degradation
Addition of poly A tail
RNA splicing
Introns are removed by spliceosomes
RNA splice sites & spliceosomes
Branch site
Overview of RNA processing in eukaryotes!
Don t forget RNA processing
before leaving the nucleus
Cap - Splicing - Tail
Overview of RNA product
(Leader)
(Trailer)
Nuclear pore structure and function
Overview of transcription and gene
expression
Our genome is extremely large, what
distinguishes specific RNA polymerase
initiation sites within it?
The PROMOTER is the DNA region that
dictates where transcription initiates
Gene Regions
TRANSCRIPTION
INITIATION
PROMOTER
ATG
START
CODON
GENE
TRANSCRIPTION
TERMINATION
TAA
TAG
TGA
TERMINATOR
STOP
CODONS
Promoter Region!
Enhancer
Promoter-proximal region
Promoter
TATA
Box
(-30 bp)
Start
point
Template
DNA strand
Binding sites for various
transcription factors
ENHANCERS
ENHANCERS are bound specifically by Transcription
factors that specify the time and place of TRANSCRIPTION
Enhanceosome
Multiprotein complexes form on enhancers
Enhancers & enhanceosomes increase
the strength of promoters!
Upstream Promoter Elements
CCAAT box
Found in selected promoters
Transcription factors bind:
CCAAT-binding transcription factor (CTF)
CCAAT/enhancer binding protein (C/EBP)
Upstream Promoter Elements
GC boxes
Variety of promoters, usually upstream of
TATA box; not all promoters
GGGCGG, CCGCCC
Show some position, orientation
independence
Need to be close to transcription start site differ from enhancers
Specific transcription factors bind, aid RNA
polymerase II binding
Role of TATA Box
Delete TATA box: transcription still occurs, but
no longer begins at specific site
TATA box often regulates transcription start site
rather than amount of transcript
Delete between TATA box and normal
transcription start point; transcription starts at
same place relative to TATA box
In some promoters, deleting TATA box
severely reduces transcription efficiency
Promoting sequences highly conserved
within promoters of eukaryotic DNA
TATA and CAAT boxes are located at about the same positions in most
promoters. The GC and octamer boxes may or may not be present; when
present, they occur at different location, either singly or in multiple copies.
Goals and timing of DNA and RNA synthesis are very different
RNA synthesis is very dynamically regulated
Making many copies is a way to amplify the information present in DNA
The conserved C-terminal domain of TBP
binds to TATA-box DNA!
TBP is a
subunit of
TFIID!
RNA and Transcription
