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Chapter 12 and 13: Transcription and Translation
Lecture 12 October 28, 2003
What’s due?
CH6 and CH10 problem set (if you haven’t all ready turned it in)
CH 11 problem set
Review: Molecular Basis of Genetics, so far…
Structure
•DNA as the genetic material
*Griffith – “transforming principle”
*Avery, MacLeod and McCarty - DNA was the “transforming principle”
*Hershey and Chase - DNA was the genetic material
•Structural analysis of DNA
*Composed of nucleotides –
deoxyribose
phosphate group
nitrogenous base
*Strands are antiparallel
and complementary
A – T
C - G
Review: Molecular Basis of Genetics, so far…
Replication
•Mode of DNA Replication
*Meselson and Stahl – “heavy” and
“light” nitrogen isotopes
•Semiconservative - each DNA molecule
consists of one parental and one newly
synthesized strand
•Origin of replication
•Bi-directional
•Roles of each polymerase (prokaryotes):
DNA polymerase I - primer removal, gap-filling synthesis
DNA polymerase II - DNA repair
DNA polymerase III - main replication enzyme
•At least six DNA polymerases in eukaryotes
Review: A Coherent Model of DNA Replication
•Helicases unwind helix (DnaA, B and C)
•SSBPs prevent closure
•DNA gyrase reduces tension
•Association of core polymerase with template
•Primase synthesizes short RNA primer
•DNA synthesis (DNA pol III)
•Primer removal and replacement with DNA (DNA pol I)
•Ligase closes up the gaps b/w Okazaki fragments
Gene Expression: Transcription and Translation
Gene expression – mechanism by which hereditary factors are coded for
and expressed (“to cause a gene to manifest its effects in the
phenotype” or “the detectable effect of a gene”)
Gene – unit of inheritance which occupies a specific
chromosomal location
Also...
TEXT: A DNA sequence coding for a single polypeptide
KSM: A DNA sequence that produces a functional RNA molecule
Type of RNA
Encodes
Copies/genome
mRNA
Functional protein
Single or few
tRNA
Molecule needed for translation
Few
rRNA
Component of ribosomes
Many
*Non (protein) coding RNA’s
Gene Expression
Protein coding gene - A DNA sequence coding for a single polypeptide
Gene expression – mechanism by which
hereditary factors are coded for and
expressed
Genes control inherited variation via:
DNA, RNA and protein
*Transcription – transfer of genetic
information from DNA via synthesis
of RNA
*Translation– the formation of a
protein, directed by an mRNA in
association with a ribosome
Phenotype
Gene: A Molecular Description
RNA Transcript
terminus
+1 start site
5’
3’
3’
5’
Coding Region
Promoter
5’ UTR
3’ UTR
Coding region – contains nucleotide sequence that encodes a specific
protein product (this region will be translated)
In eukaryotes: introns and exons
Non-coding regions – contains nucleotide sequence that will get
transcribed BUT not translated
*Un-translated regions (UTR’s)
Promoter regions – sequence involved in the control of expression of a
given gene, site where RNA polymerase binds
Regulatory regions – sequence involved in the control of expression of
a given gene, usually involves interaction with another molecule
Gene: A Molecular Description
Only one of the two strands encodes the mRNA for a given gene
Template strand – coding strand – sense strand = template
for transcription
Non-template strand – nonsense strand = RNA transcript is
exactly the same as the non-sense strand
5’
A A A G T C C G G T A C G 3’
3’ T
Coding strand
T T C A G G C C A T G C 5’
Given that RNA polymerase synthesizes RNA in a 5’ to 3’ direction,
which strand is the template strand?
3’ U
U U C A G G C C A U G C 5’
*Transcript will always “look” like the non-sense strand
Transcription
Transcription – the process by which RNA molecules are synthesized on
a DNA template
*RNA polymerase – enzyme that copies template strand to build an
RNA molecule
-synthesis in 5’ to 3’ direction
–nucleotides added to 3’-OH
–growing strand has base complementarity to template strand
–unlike DNA pol, no primer required
reminder: RNA contains ribose, phosphate group and A, C, G and U (not T)
*RNA polymerase (from E. coli )
s a2 b b’
Sigma factor
Core
Holoenzyme
Sigma factor – helps drive the
polymerase to the promotor
Core – responsible for elongation
Holoenzyme responsible for initiation =
binding of the polymerase to the promotor
Transcription
Factor – something that cycles on and off core complexes
Multiple types of sigma factors in bacterial cells - regulation
s1
P1
s2
P2
s3
P3
Promotors - sequence involved in the control of expression of a given
gene, site where RNA polymerase binds
+1 start site
5’
3’ TTGACA
TATAAT
-35 region
RNA Transcript
Coding Region
-10, TATA box, Pribnow box
~17 base spacer
Serve three different functions:
1. ON/OFF switch
2. “Speed” switch
3. Alignment
3’
5’
Transcription in Eukaryotes
*RNA polymerases:
RNA polymerase I – rRNA (18S, 28S)
RNA polymerase II – mRNA
RNA polymerase III – small RNA’s ( tRNA, 5S rRNA, snRNA’s)
Eukaryotic promotors:
CAAT box
Hogness box
CAAT box
Hogness box
Hogness box
CAAT box
Goldberg-Hogness box, TATA box, -25 (all)
CAAT box, -80 (many)
Enhancers
Hogness box
Transcription in Eukaryotes
“Generalized Transcription Factors”–group of proteins that bind the -25 region
Transcription Factor for RNA polymerase II – TFIIA, TFIIB, etc.
*TFII’s – not enough! Need factors that bind -80 and enhancers
1. ON/OFF switch = -25 region
2. “Speed” switch = enhancers
Elongation – very similar in prokaryotes and eukaryotes
Termination -Transcription stops - Polymerase and RNA are released
from DNA - DNA rehybdridizes
RNA processing in Eukaryotes
Immature RNA – mature RNA
*Addition of a cap at 5’ end- guanyltransferase – makes mRNA more
stable, required for translation
*Addition of a poly A tail – poly A polymerase – mRNA stability, translation
*Introns spliced out by spliceosome machinery