Transcript Protein synthesis

Make a copy of DNA (in nucleus)
 Send copy out of nucleus into cytoplasm
 Read copy on ribosome
 Make a protein


“Central Dogma”
Nucleus & Nucleolus
 Ribosomes
 Endoplasmic Reticulum
 Golgi Apparatus
 Vesicles


Flow of genetic information in a cell
 How do we move information from DNA to proteins?
Replication
Goal
Make a copy of DNA in
the form of RNA
Location
NUCLEUS

Transcription Unit (GENE): segment of DNA to be
transcribed
 transcribed DNA strand = Template Strand
 Only read1 strand (template strand)…make complimentary
mRNA strand

transcription bubble
 enzyme
○ RNA Polymerase
5
A
G
T
A T C
T A
C
3
G
G
coding strand
A G C
C
T
A
T
C G T
A
T
3
A
G C A U C G U
C
G T A G C A
T
C
A G
C T
G
A
T
A
T
3
5
unwinding
rewinding
mRNA
5
RNA polymerase
template strand
Promoter: identified by RNA poly…attach
 Gene: actual DNA turned into mRNA
 Terminator: RNA poly detaches


Eukaryotes
 Transcription Factors
○ Protein helpers
○ Help RNA poly attach to
promoter
○ Looks for TATA BOX
○ TATA Box
 Upstream end of
promoter region
RNA Polymerase
moves along DNA,
opening 10-20 bases
at a time
 Adds new RNA Nt to
the growing 3’ end
 RNA strand peels
away & DNA helix
reforms

reads DNA 35

Animations:
 http://www-
class.unl.edu/bioche
m/gp2/m_biology/an
imation/gene/gene_a
2.html
 http://vcell.ndsu.edu
/animations/transcri
ption/movieflash.htm
What would be the
complementary RNA strand
for the following DNA
sequence?
DNA
-GCGTATG-

Eukaryotes
 RNA Polymerase
transcribes a
“signal” (AAUAAA)
in terminator
region of gene
 END
mRNA
Goal
Read mRNA and turn
into a PROTEIN
Ribosome
Goal
RIBOSOME (cyto or
RER)

Structure
 ribosomal RNA (rRNA)
& proteins
 2 subunits
○ large
○ small

A site (accepting site)
 holds tRNA carrying next
amino acid to be added
to chain

P site (protein syn site)
 holds tRNA carrying
growing polypeptide
chain

E site (exit site)
 empty tRNA
leaves ribosome
from exit site
mRNA is read in sets
of 3 bases…CODON
 CODON codes for
Amino Acid



Start codon
•
•
AUG
methionine
•
UGA, UAA, UAG
Stop codons
start
codon
mRNA
A U G G G C U C C A U C G G C G C A U A A
codon 1
protein
methionine
codon 2
codon 3
glycine
serine
codon 4
isoleucine
codon 5
codon 6
glycine
alanine
codon 7
stop
codon
Primary structure of a protein
aa1
aa2
aa3
peptide bonds
aa4
aa5
aa6

Initiation
 brings together mRNA, ribosome
subunits, initiator tRNA

Elongation
 adding amino acids based on codon
sequence

Termination
 stop codon
3 2 1
Leu
Val
Met
Met
Met
Met Leu
Ala
Leu
Leu
release
factor
Ser
Trp
tRNA
U AC
5'
C UGAA U
mRNA A U G
3'
E P A
5'
UAC GAC
A U G C U GAA U
5'
3'
U A C GA C
A U G C U G AAU
5'
3'
U AC G A C
AA U
AU G C U G
3'
A CC
U GG U A A
3'
1.
Small ribosomal subunit binds to mRNA
○ Start codon AUG= methionine AA
2.
Large ribosomal subunit binds
○ Closes down on small
1.
tRNA brings in A.A and it
sits in A site
2.
Enzyme in the large
subunit of the ribosome
catalyzes a peptide bond
between new AA in A
site and AA in P site
3.
Ribsome moves the A
site tRNA to the P site

Empty tRNA at P site
ejected from E site
Stop codon reaches A site
 Release factors bind to A site
 Polypeptide released


http://vcell.ndsu.edu
/animations/translati
on/movie-flash.htm
What would be the complementary
mRNA strand and amino acid
sequence for the following DNA
mRNA: 5’AGCAUG-CCC-UAU-GGG-UUU-AUA-GCU-UGAGAG
3’
sequence?
tRNA anticodons: UAC-GGG-AUA-CCC-AAA-UAU-CGA-ACU
Polypeptide: met-pro-tyr-gly-phe-ile-ala
DNA
-TCGTACGGGATACCCAAATATCGAACTCTC-

“Clover leaf” structure
 Anticodon on “clover leaf” end
○ Complementary to mRNA codon
 Amino Acid attached on 3 end

Transcription initiation: RNA Polymerase recognizes
promoter and binds…no transcription factors needed

3 RNA polymerase enzymes
 RNA polymerase 1
○ only transcribes rRNA genes
○ makes ribosomes
 RNA polymerase 2
○ transcribes genes into mRNA
 RNA polymerase 3
○ only transcribes tRNA genes

each has a specific promoter
sequence it recognizes

Transcription & translation are simultaneous in
bacteria
 DNA is in
cytoplasm
 no mRNA
editing
 ribosomes
read mRNA
as it is being
transcribed

2 categories:
 Base – pair substitution
 Base – pair insertions or deletions

Replacement of one nucleotide
 Silent mutations: do not
present a change in protein
○ (multiple codons for one
amino acid)
 Missense mutations: still
code for an amino acid; but
the wrong amino acid.
 Nonsense mutation: codes
for a STOP CODON –
translation ends prematurely

Additions or losses of
nucleotide pairs in a
gene
 Have deleterious
effects b/c alter the
“reading frame” of the
genetic message =
frameshift mutation
○ Occurs whenever
the insertion or
deletion is NOT a
multiple of three

Histone acetylation turns genes = ON
 attachment of acetyl groups (–COCH3) to positively charged lysines
(neutralize AA)
○ when histones are acetylated they change shape & grip DNA
less tightly = unwinding DNA
○ transcription proteins have easier access to genes
Chromatin modifications affect the
availability of genes for
transcription
 DNA methylation turns genes = off

 attachment of methyl groups (–CH3) to
DNA bases (cytosine) after DNA is
synthesized
○ nearly permanent suppression of genes
○ ex. the inactivated mammalian X
chromosome

Primary transcript (pre-mRNA)
 eukaryotic mRNA needs work after transcription

mRNA processing (making mature mRNA)
 mRNA splicing = edit out introns (non-coding regions)
 protect mRNA from enzymes in cytoplasm
○ add 5’ G cap
3'
○ add 3’ Poly A tail
mRNA
A
PP
5' G P
intron = noncoding (inbetween) sequence
~10,000 bases
eukaryotic DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
mature mRNA
transcript
~1,000 bases
spliced mRNA

snRNPs
 small nuclear RNA
 proteins

snRNPs
snRNA
intron
exon
exon
5'
Splicesome
3'
 several snRNPs
 recognize splice site
sequence
○ cut & paste
spliceosome
5'
3'
lariat
No,
not smurfs!
“sNurps”
mature mRNA
5'
exon
5'
3'
exon
3'
excised
intron

http://vcell.nds
u.edu/animatio
ns/mrnaprocess
ing/movieflash.htm