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Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
RNA Polymerase
RNA polymerase in E. coli consists of 4 different subunits (see model below).
s recognizes the promoter. Holoenzyme is needed for correct initiation of transcription.
RNA polymerase adds ribonucleotides in 5’ to 3’ direction.
A single type of RNA polymerase transcribes RNA in prokaryotes.
Promoter Sequences in E. coli
Promoters signal transcription in prokaryotes.
Transcription Initiation in Prokaryotes
s subunit positions RNA
polymerase for correct
initiation.
Upon initiation of transcription,
s subunit dissociates.
Elongation
RNA polymerase adds ribonucleotides in 5’ to 3’ direction.
RNA polymerase catalyzes the following reaction:
DNA
NTP + (NMP)n
(NMP)n+1 + PPi
Mg++
RNA polymerase
Termination
Termination of transcription occurs beyond the
coding sequence of a gene. This region is 3’
untranslated region (3’ UTR), which is recognized by
RNA polymerase.
Termination
RNA polymerase recognizes signals for chain termination.
(1) Intrinsic: Termination site on
template DNA consists of GC-rich
sequences followed by A’s. Intramolecular hydrogen bonding causes
formation of hairpin loop.
(2) rho factor (hexameric protein) dependent:
These termination signals do not produce
hairpin loops. rho binds to RNA at rut site.
rho pulls RNA away from RNA polymerase.
rut site
In E. coli, this structure signals
release of RNA polymerase, thus
terminating transcription.
Colinearity of Gene and Protein
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Genetic Code
Genetic Code is nonoverlapping.
A codon (three bases or triplet)
encodes an amino acid.
Genetic Code is read
continuously from a fixed
starting point.
There is a start codon (AUG).
There are three stop (termination) codons. They are often called nonsense codons.
Genetic Code is degenerate. Some amino acids are encoded by more than one
codon.
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Eukaryotic RNA
Three RNA Polymerases
RNA Polymerase
Synthesis of
I
II
III
rRNA (except 5S rRNA)
mRNA*, some snRNA
tRNA, some snRNA, 5S rRNA
* eukaryotic RNA is monocistronic
prokaryotic RNA can be polycistronic
Eukaryotic RNA
Many proteins must assemble at
promoter before transcription.
General transcription factors
(GTF’s) bind before RNA
polymerase II, while other proteins
bind after RNA polymerase II binds.
Primary transcript (pre-mRNA) must
be processed into mature mRNA.
Chromatin structure affects gene
expression (gene transcription) in
eukaryotes.
1. Cap at 5’ end (7-methylguanosine)
2. Addition of poly(A) tail
3. Splicing of RNA transcript
Prokaryotic and Eukaryotic Transcription and Translation Compared
Transcription Initiation
in Eukaryotes
TATA binding protein (TBP),
part of TFIID complex, must
bind to promoter before
other GTFs and RNA
polymerase II can form
preinitiation complex (PIC).
Phosphorylation of carboxyl
tail domain (CTD), the
protein tail of b subunit of
RNA polymerase II, allows
separation of RNA
polymerase II from GTFs to
start transcription.
Cotranscriptional Processing
of RNA
State of phosphorylation of
CTD determines the type of
proteins that can associate with
the CTD (thus defining
cotranscriptional process).
5’ end of pre-mRNA is capped
with 7-methylguanosine. This
protects the transcript from
degradation; capping is also
necessary for translation of
mature mRNA.
Cotranscriptional Processing
3’ end of the transcript typically contains
AAUAAA or AUUAAA.
This sequence is recognized by an enzyme that
cleaves the newly synthesized transcript ~20
nucleotides downstream.
At the 3’ end, a poly(A) tail consisting of 150 200 adenine nucleotides is added.
Polyadenylation is another characteristic of
transcription in eukaryotes.
Complex Patterns of Eukaryotic RNA Splicing
Different mRNA can be produced; different a-tropomyosin can be produced.
Alternative splicing is a mechanism for gene regulation. Gene product can be different
in different cell types and at different stages of development.
Intron Splicing: Conserved Sequences
exons - coding sequences
introns - noncoding sequences
Small nuclear ribonucleoprotein particles (snRNPs) recognize
consensus splice junction sequence of GU/AG.
snRNPs are complexes of protein and small nuclear RNA (snRNA).
Several snRNPs comprise a spliceosome.
Spliceosome directs the removal of introns and joining of exons.
Spliceosome Assembly
and Function
Spliceosome interacts
with CTD and attaches to
pre-mRNA.
snRNAs in spliceosomes
direct alignment of the
splice sites.
One end of conserved
sequence attaches to
conserved adenine in
the intron.
The “lariat” is released
and adjacent exons are
joined.
Reactions in
Exon Splicing
Self-Splicing Reaction
RNA molecules can act
somewhat like enzymes
(ribozymes).
In the protozoan
Tetrahymena, the primary
transcript of an rRNA can
excise a 413-nucleotide
intron from itself.
These self-splicing
introns are an example
of RNA that can catalyze
a reaction.