Transcript Lecture16updated Biol302 Spring 2011

What does the word Promoter mean?
It is the place at which RNA Pol II binds.
But the word is incorrectly used to describe
Enhancers plus Promoter.
Initiation by RNA Polymerase II
TFIID recognition site is TATAA
How often is this site found in the
genome? 1/45
Once every 1000 nucleotides 109 nucleotides or 106 times
Transient transfection
More Cells
But on a per cell
Basis expression levels of -gal
is about the same
Stable transfection
Recruitment Model
The 7-Methyl Guanosine
(7-MG) Cap
The 3’ Poly(A) Tail
AATAAA
Interrupted Genes in Eukaryotes:
Exons and Introns
Most eukaryotic genes contain noncoding sequences called
introns that interrupt the coding sequences, or exons. The introns
are excised from the RNA transcripts prior to their transport to the
cytoplasm.
Removal of Intron Sequences
by RNA Splicing
The noncoding introns are excised
from gene transcripts by several
different mechanisms.
Excision of Intron Sequences
Splicing
 Removal of introns must be very precise.
 Conserved sequences for removal of the introns of
nuclear mRNA genes are minimal.
– Dinucleotide sequences at the 5’ and 3’ ends of introns.
– An A residue about 30 nucleotides upstream from the 3’
splice site is needed for lariat formation.
Types of Intron Excision
 The introns of tRNA precursors are excised by precise
endonucleolytic cleavage and ligation reactions
catalyzed by special splicing endonuclease and ligase
activities.
 The introns of nuclear pre-mRNA (hnRNA) transcripts
are spliced out in two-step reactions carried out by
spliceosomes.
The Spliceosome
 Five snRNAs: U1, U2, U4, U5,
and U6
 Some snRNAs associate with
proteins to form snRNAs (small
nuclear ribonucleoproteins)
What are Logo plots?
Logo for
a) Splice acceptor
b) Splice Donor
c) Initiator Met
AG/GT
exon 1
CAG/NT
intron 1
exon 2
Chapter 12
Translation and the Genetic Code
Protein Structure
Proteins are complex
macromolecules
composed of 20 (?)
different amino acids.
Amino Acids
Proteins are made of polypeptides.
A polypeptide is a long chain of amino acids.
Amino acids have a free amino group, a free
carboxyl group, and a side group (R).
Peptide Bonds
Amino acids are joined by peptide bonds.
The carboxyl group of one amino acid is
covalently attached to the amino group of the
next amino acid.
Protein Synthesis: Translation
The genetic information in mRNA molecules is
translated into the amino acid sequences of
polypeptides according to the specifications of
the genetic code.
The Macromolecules of
Translation
Polypeptides and rRNA molecules
Euk: 28S, 18S, 5.8S, 5S
Amino-acid Activating Enzymes
tRNA Molecules
Soluble proteins involved in polypeptide
chain initiation, elongation, and
termination
Why does one need the ribosome to translate mRNA?
Ribosomes
What does “S” mean?
Why do sizes get bigger?
The Nucleolus
In eukaryotes, the
nucleolus is the site
of rRNA synthesis
and ribosome
assembly
Synthesis and Processing of the
30S rRNA Precursor in E. coli
ProKaryote
numbers
Synthesis and Processing of the
45S rRNA Precursor in Mammals
rRNA Genes
rRNA Genes in E. coli
– Seven rRNA genes distributed among three sites
on the chromosome
rRNA Genes in Eukaryotes
– rRNA genes are present in hundreds to thousands
of copies
– The 5.8S-18S-28S rRNA genes are present in
tandem arrays in the nucleolar organizer regions
of the chromosomes.
– The 5S rRNA genes are distributed over several
chromosomes.
Transfer RNAs (tRNAs)
 tRNAs are adapters
between amino acids and
the codons in mRNA
molecules.
 The anticodon of the
tRNA base pairs with the
codon of mRNA.
 The amino acid is
covalently attached to the
3’ end of the tRNA.
 tRNAs often contain
modified nucleosides.
What is Inosine?
Inosine
tRNA Structure
Specificity of tRNAs
tRNA molecules must have the correct
anticodon sequence.
tRNA molecules must be recognized by
the correct aminoacyl-tRNA synthetase.
tRNA molecules must bind to the
appropriate sites on the ribosomes.
Codon Specificity Resides in the tRNA,
Not the Attached Amino Acid.
tRNA Binding Sites on the
Ribosome (Ribosme moves
like an enzyme)
Stages of Translation
Polypeptide Chain Initiation
Chain Elongation
Chain Termination
Translation Initiation in E. coli
30S subunit of the ribosome
Initiator tRNA (tRNAMet)
mRNA
Initiation Factors IF-1, IF-2, and IF-3
One molecule of GTP
50S subunit of the ribosome
The Shine-Dalgarno
Sequence
Translation Initiation in
Eukaryotes
The amino group of the methionine on the
initiator tRNA is not formylated.
The initiation complex forms at the 5’ terminus
of the mRNA, not at the Shine-Dalgarno/AUG
translation start site.
The initiation complex scans the mRNA for an
AUG initiation codon. Translation usually
begins at the first AUG.
Kozak’s Rules describe the optimal sequence
for efficient translation initiation in eukaryotes.
Polypeptide Chain Elongation
An aminoacyl-tRNA binds to the A site of the
ribosome.
The growing polypeptide chain is transferred
from the tRNA in the P site to the tRNA in the
A site by the formation of a new peptide bond.
The ribosome translocates along the mRNA
to position the next codon in the A site. At the
same time,
– The nascent polypeptide-tRNA is translocated
from the A site to the P site.
– The uncharged tRNA is translocated from the P
site to the E site.
Elongation of Fibroin
Polypeptides (A mRNA can
have multiple Ribosomes
Polypeptide Chain
Termination
Polypeptide chain termination occurs when a
chain-termination codon (stop codon) enters
the A site of the ribosome.
The stop codons are UAA, UAG, and UGA.
When a stop codon is encountered, a release
factor binds to the A site.
A water molecule is added to the carboxyl
terminus of the nascent polypeptide, causing
termination.
No tRNA exists for stop codons!
Dissociation upon finish of protein synthesis
The Genetic Code
The genetic code is a nonoverlapping
code, with each amino acid plus
polypeptide initiation and termination
specified by RNA codons composed of
three nucleotides.
Properties of the Genetic Code
The genetic code is composed of nucleotide
triplets.
The genetic code is nonoverlapping. (?)
The genetic code is comma-free. (?)
The genetic code is degenerate. (yes)
The genetic code is ordered. (5’ to 3’)
The genetic code contains start and stop
codons. (yes)
The genetic code is nearly universal. YES :)
A Triplet Code*
A Single-Base Pair Insertion
Alters the Reading Frame*
A suppressor mutation restores
the original reading frame.*
Insertion of 3 base pairs does
not change the reading
frame.*
Evidence of a Triplet Code:
In Vitro Translation Studies
Trinucleotides were sufficient to stimulate
specific binding of aminoacyl-tRNAs to
ribosomes.
Chemically synthesized mRNAs containing
repeated dinucleotide sequences directed the
synthesis of copolymers with alternating
amino acid sequences.
mRNAs with repeating trinucleotide
sequences directed the synthesis of a mixture
of three homopolymers.
Deciphering the Genetic Code
You must know single letter codes and some triplets!
The Genetic Code
Initiation and termination Codons
– Initiation codon: AUG
– Termination codons: UAA, UAG, UGA
Degeneracy: partial and complete
Ordered
Nearly Universal (exceptions:
mitochondria and some protozoa)
Key Points
Each of the 20 amino acids in proteins is
specified by one or more nucleotide triplets in
mRNA. (20 amino acids refers to what is
attached to the tRNAs!)
Of the 64 possible triplets, given the four
bases in mRNA, 61 specify amino acids and
3 signal chain termination. (have no tRNAs!)
Key Points
The code is nonoverlapping, with each
nucleotide part of a single codon, degenerate,
with most amino acids specified by two to four
codons, and ordered, with similar amino acids
specified by related codons.
The genetic code is nearly universal; with
minor exceptions, the 64 triplets have the
same meaning in all organisms. (this is funny)
Do all cells/animals make the same
Repertoire of tRNAs?
The Wobble Hypothesis:
Base-Pairing Involving the Third
Base of the Codon is Less Stringent.
Base-Pairing with Inosine at
the Wobble Position
Suppressor Mutations
Some mutations in tRNA genes alter the
anticodons and therefore the codons
recognized by the mutant tRNAs.
These mutations were initially detected as
suppressor mutations that suppressed the
effects of other mutations.
Example: tRNA mutations that suppress
amber mutations (UAG chain-termination
mutations) in the coding sequence of genes.
Making a (UAG) Mutation
Translation of an amber (UAG)
Mutation in the Absence of a
Suppressor tRNA
Translation of an amber Mutation in
the Presence of a Suppressor tRNA
Note it is amber su3…why?????????
Translation of an amber Mutation in
the Presence of a Suppressor tRNA
If there was a single tRNATyr gene, then could one
have a amber supressor of it?
Historical Comparisons
Comparison of the amino acid sequence of
bacteriophage MS2 coat protein and the
nucleotide sequence of the gene encoding
the protein (Walter Fiers, 1972).
Was this first????
Sickle-cell anemia: comparison of the
sequence of the normal and sickle-cell alleles
at the amino acid level and at the nucleotide
level.
Are the proteins produced a
pure reflection of the mRNA
sequence????
tRNA environment, protein modifications post-translationally
To Know for Exam
RNApol II
TATAA
CCATGG (Nco I site and Kozak Rule)
ATG
AGGT….splice
GT……………A………polypyrimidine AG
PolyA recog sequence
AATAAA
The Reasons why ATG is a single codon
and TGG is a single codon.