9.1-9.2 Flashcards
Genetic code is the language that allow
DNA and RNA sequences to be
translated into proteins
Two nucleotides
4^2 = 16 possible codons
how many amino acids
20
codon
nucleotide triplet
code is partically
redunant more than one codon per given AA
Charles Yanofsky and Sydney Brenne
in 1960s, collected evidence that supported gene-protein colinearity
1. The length of the gene is proportional to the length of the protein
2.Consecutive nucleotides in a gene from the start to stop determine the consecutive/linear order of amino acids in a protein
Yanofsky and his lab generated a set
Trp- auxotrophic mutants in E. coli
Trp- auxotrophic mutants in E. coli
Mutations in trpA gene, encoding a subunit of the enzyme tryptophan synthase
Trp- auxotrophic mutants in E. coli
Mutations in trpA gene, encoding a subunit of the enzyme tryptophan synthase
Yanofsky was the first to
Created a fine-structure recombination map of these mutations using P1 bacteriophage and determined the amino acid sequence of the mutant tryptophan synthase
Point mutations altering different nucleotides may affect the
SAME amino acid (missense mutations)
A Gene’s Nucleotide Sequence is
Colinear with the Amino Acid Sequence of the Encoded Polypeptide
Francis Crick and Sydney Brenner
In 1955, used bacteriophage T4 rIIB gene with Proflavin mutation
Missense mutation
mutation in gene that changes a codon for one amino acid to a codon that specifies a different amino acid
Yanofsky observed
missense mutation
Francis Crick and Sydney Brenner proved
codons are 3 nuclotides
proflavin molecules cause
single base insertions or deletions (frameshift mutation)
In vitro Translation System
cellular extracts that upon addition of mRNA
can lead to polypeptide synthesis in a test tube
Marshall Nirenberg and Heinrich Matthaei
In 1961, added a synthetic poly-U (5’…
UUUUUUUUUU…3’) mRNA to cell-free
translational system derived from E.coli.
deciphering the genetic code
Nirenberg, Khorana and Holley
In 1965, Nirenberg and Philip Leder
added short synthetic mRNA ONLY 3
nucleotides in length to an in vitro translational
system containing tRNA attached to amino acids,
where only 1 of the 20 amino acids was
radioactive
Nirenberg and Philip Leder
Codon-amino acid correspondences
“STOP” codons
UGA, UAA and UAG
Sydney Brenner
indentified stop codons
Sydney Brenner used
Point mutations in T4 phage head protein “m”, encoding a component
of phage head capsule
nonsense mutation
changes a codon
that signifies an amino acid (a sense
codon) into one that does not (STOP CODON)
5’ to 3’ in mRNA corresponds to
N to C-terminus in the polypeptide
Proteins are encoded by
non-overlapping triplets of nucleotides called codons in a given
gene
initiation codon, AUG
codes for methionine at the start of the
reading frame
The genetic code is
degenerate
The three stages of transcription
- Initiation
- Elongation
- Termination
Transcription is carried out by
RNA polymerases
Initation
DNA sequences at the beginning of genes called “promoters” direct the exact location for the initation of transcription by RNA polymerase
What is required for initiation
sigma factor
Core enzyme + sigma =
holoenzyme (RNA polymerase)
sigma factor
reduces RNA polymerase’s general affinity for DNA but increases the enzyes affinity for the promoter (binds tighty) and forms closed promoter complex
Open promoter complex
RNA polyerase and unwound promoter
Elongation
Constructing an RNA copy of the gene RNA poly slides along the DNA to synthesize RNA
Elongation rate
50 nucleotides per sec
Once an RNA poly moves way from promoter
a second RNA poly can bind the promoter and initiate transcription
A can express different genes at
different rates
Termination
a terminator is reached that RNA poly and RNA transcipt to dissociate from DNA
Intrinsic terminators
cause RNA poly to terminate transciption on its own
Extrinsic terminators
require addition proteins (Rho protein)
Transciption has many
initiator sites
Transcription uses ___ for energy
NTPs
Only small portion of DNA is
transcipted
AIDS uses
reverse transciption
eukaryotic RNA poly II
transcribes genes that encode proteins
reverse transcription
reverse transciptase synthesizes DNAstrands complementary to an RNA template
reverse transcription product
cDNA
RNA processing
in eukaryotes, converts RNA into mRNA
Modification for mRNA
splicing exons (removing introns), and addition of a poly-A tail to the 3’ end and a methylated cap at the 5’ end
Methylated cap
crucial for efficient translation of mRNA to protein
poly-A tail
consisting of 100-200 A resiues, that stabilizes the mRNA and increases the efficiency of translation initiation
Eukaryotes use ___ that bind to protein factors aiding transciption
enhancer
basal transcription
core promoter by itself produces low level of transcription
Regulatory elements
affect the binding of RNA poly to the promoter
1. Enhancer: stim. transcription
2. Silencers: Inhibit transciption
RNA poly I
rRNA
RNA poly III
tRNA
Methy transferases
add methy (-CH3) groups to backward G
m7G meth cap
Poly-A tail length
100-200 As long
Translation initiation factors bind to
methylated cap, while poly-A binding
proteins associate with the 3’ poly-A tail
Enhances translation
initiation
DNA nucleotide seq of many eukaryotic
genes are much longer than corresponding
mRNA
we remove introns
human gene DMD
encodes the protein Dystrophin and mutation can cause Duchenne Muscular Dystrophy
Exons (for expressed regions):
Sequences found in both a gene’s DNA
and mature mRNA
RNA Splicing:
The process that deletes introns and
joins together successive exons to
form mature RNA
Splicing requires a complicated intranuclear machine
Spliceosome
Spliceosome consists of four subunits called
Small nuclear ribonucleoproteins OR snRNPS
Each snRNP contains
1 or 2 small nuclear RNAs
(snRNAs
snRNP
-100-300 nucleotides long associated
with proteins in discrete particle.
Splicing is catalyzed by
splicesome
Ribozymes
RNA molecules that can act as enzymes to catalyze specific reactions (splice themselves) have 5 snRNAs
alternative splicing
production of different mature mRNAs from the same primary RNA by joining different combinations of exons
Alternative splicing largely explains how
28,000 genes in the human genome can encode hundred of thousands different proteins
Alternative splicing can regulate
localization of proteins and their enzymatic properties.
isoforms
Proteins resulting from alternative splicing
Introns allow for
alternative splicing
Introns can generate
non-coding RNAs (ncRNA) that influence gene expression
snRNA
components of slocesome which is required for RNA splicing
