Molecular Biology
Transcription &
Translation
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Researchers

Archibald Garrod (1909)


genes dictate phenotypes
diseases are “inborn errors of metabolism” (inability
to make particular enzyme)
Researchers, cont.


George Beadle and Edward Tatum
worked with bread mold applying X-rays looking
for mutations


one gene-one enzyme hypothesis
Not all gene products are enzymes, restated

one gene-one polypeptide hypothesis
Expressing the Genotype



bridge between genotype and phenotype is a
protein
genes give instructions for making specific
proteins, but they do not make it directly
gene (DNA) RNA
polypeptide
Transcription



DNA to RNA
reading 5’ to 3'
in nucleus
RNA Types

messenger


transfer


tRNA anticodons, carries amino acids
ribosomal


mRNA codons, processed version
rRNA protein synthesis
primary transcript (pre-mRNA)

initial RNA transcript from any gene
Protein Synthesis

Transcription


nucleus
Translation

cytoplasm
Transcription DNA
DNA
A
T
C
G
RNA
RNA (complementary bases)
U
A
G
C
Eukaryotes
transcription

DNA



pre-mRNA
Initiation
elongation
termination

promoter: DNA sequence where transcription starts
terminator site: signals the end of transcription
transcription unit: stretch of DNA that is transcribed
coding strand: makes sense, is the template

noncoding strand: makes no sense, not the template



Initiation of Transcription in Eukaryotes
INSIDE NUCLEUS
promoter
TATA Box
transcription factors
promoter determines which strand of DNA will be
the template, it is also the binding site for RNA
polymerase
TATA Box nucleotide sequence including T-A-T-A
is present in the promoter
Transcription factors recognize the TATA box and
mediate the binding to the DNA
RNA polymerase II
Additional transcription factors bind to the DNA
along with the RNA polymerase II forming the
transcription initiation complex. DNA unwinds
and RNA synthesis begins.
RNA transcription
Elongation
DNA unwinds one turn
at a time, 10 to 20
bases at time
attach RNA nucleotides
to DNA template
double helix re-forms
and the RNA molecule
separates
Termination of Transcription


Bacteria: specific DNA sequence indicates end of
production of mRNA, polymerase detaches
Eukaryotes: more complex



special sequence is coded (AAUAAA)
after the passing of the sequence the pre-mRNA is let
free
RNA polymerase keeps coding while digestive
enzyme digests its product until it reaches the
polymerase, then the polymerase detaches
Modifications to pre-mRNA


to prevent degradation
cap and tail




add 5’ cap (guanosine triphosphate)
add 3’ poly A tail (50-250)
nontranslated leader and trailer segments
in nucleus
RNA Splicing
pre-mRNA

pre-mRNA



mRNA
introns - intervening (noncoding) sequences
exons - expressing (coding) sequences
mRNA with exons only
Pre-mRNA
Splicing

Pre-mRNA combines with
snRNPs (“snurps”) and other
proteins to form a spliceosome

Within the spliceosome, snRNA
base-pairs with nucleotides at
the ends of the intron.

The RNA transcript is cut to
release the intron, and the exons
are spliced together; the
spliceosome then comes apart,
releasing mRNA, which now
contains only exons
(assembly line)
snurps= small nuclear ribonucleoproteins
Translation
mRNA
polypeptide


in cytoplasm
Chemical Language (U,A,C,G)
41 = 4
42 = 16
43 = 64

one-letter code:
two-letter code:
three-letter code:

triplet code specifies certain amino acids.


Reading Frame


3 base sequence
known as codon
start at 5’ end of
mRNA
Genetic Code
Ribosomes

2 subunits


small and large
binding sites




A (aminoacyl-tRNA) site
P (peptidyl-tRNA) site
E (exit) site
mRNA binding site
tRNA structure
about 80 nucleotides
anticodon
attachment site at the 3’
end for an amino acid
Translation
mRNA

3 stages



initiation
elongation
termination
polypeptide
Initiation




mRNA binds to the small unit of ribosome
small unit recognizes AUG (starting codon)
codes for methionine (Met) in the P site
tRNA carries Met at 3’ end
Elongation
3. Translocation
empty tRNA moves
from P site to E site.
Same time the tRNA
with the chain moves from
A to P; starting the process
once again
1. codon recognition tRNA anticodon recognizes codon in the A
site. Hydrolysis of GTP makes it
more accurate
2. peptide bond formation
ribosomal RNA catalyzes
the formation of peptide
bond between the amino
acids in sites P and A.
Also removes tRNA in the
P site attaching the protein
chain to the tRNA in the
A site instead
GTP= guanosine tri-P
Termination



nonsense coding (stop)
 UAA, UAG, UGA
release factor
 protein that frees polypeptide promoting hydrolysis
between tRNA and P site
subunits dissociate
Polyribosome (euk. and prok.)

cluster of ribosomes


synthesize multiple copies of same polypeptide
using same mRNA
a ribosome requires less than a minute to
translate an average-sized mRNA into a
polypeptide
Redundancy of the Genetic Code

several codons code for same a.a. to allow
for errors
example: alanine
GCU, GCC, GCA, GCG

Wobble Concept


3rd N-base wobbles on tRNA
only on tRNA



Adenine can be modified to Inosine (I)
I is complementary to U, C, A
anticodon AAI complementary
to UUU, UUC, UUA (mRNA)
AI
I
I
Ribosomes

free


synthesize protein that dissolve in cytosol and
function there
bound


synthesize protein of membranes
synthesize protein secreted from cell
Mutations

Mutations are changes in the DNA base
sequence

These are caused by errors in DNA
replication or by mutagens
Point mutations

change in one base


substitutions
deletions/insertions
Substitutions

5'
3'
Replacement of one nucleotide

Silent mutation
code for the same a.a.

Missense mutation
codes for another a.a.

Nonsense mutation
codes for stop codon
3'
5'
Point Mutation Example
Sickle-cell Disease
Val - His - Leu - Thr - Pro - Glu - Glu
free hemoglobin molecules
normal O2 capacity
Val- His- Leu- Thr - Pro - Val - Glu
molecules interact and crystallize
forming a fiber, lowers O2 capacity
Deletions/Insertions
The End