Chapter 5b

The Central Dogma : Transcription and

Translation

This material is for exclusive use of BIO 121 (General Genetics) students under the
Science Department, CNSM, Mindanao State University-General Santos.
Unauthorized use of this material is prohibited.
 Learning Outcomes References

To learn about processes

involved in mRNA and protein • Klug W and Cummings M (2009).
synthesis Concepts of Genetics. 9th ed. Prentice
Hall Inc.
Learn about the processing of • Alberts B et al (2008). Molecular
the primary transcript in Biology of the Cell. 5th ed. Garland
eukaryotes prior to translation
Science Publishing, UK.
Differentiate between • Weaver RF (2012). Molecular Biology.
prokaryotic and eukaryotic 5th ed. McGraw Hill Co., New York
transcription and translation
Learn how

 Part 1:
Transcription

from
DNA nucleic acid language
to
RNA nucleic acid language






 Transcription
■ Making mRNA
◆ transcribed DNA strand = template strand
◆ untranscribed DNA strand = coding strand
■ same sequence as RNA
◆ synthesis of complementary RNA strand
■ In the transcription bubble
◆ enzyme
■ RNA polymerase

coding strand

A G C A T C G T A
3ʹ
5ʹ G
A
T
G A
T C
T
A
C A G T
C A T C T
DNA G T A 3ʹ A C T
G 5ʹ
G C A U C G U T
C unwinding
3ʹ G T A G C A
rewinding

mRNA 5ʹ RNA polymerase template strand

build RNA 5ʹ→3ʹ

 Bacterial chromosome

Transcription in Transcription
mRNA
Prokaryotes

Cell
membrane

Cell wall

 Transcription in Prokaryotes
■ Initiation
◆ RNA polymerase binds to promoter sequence
on DNA

Role of promoter
■ Starting point
◆ where to start reading
◆ start of gene

■ Template strand
◆ which strand to read

■ Direction on DNA
◆ always read DNA 3ʹ→5ʹ

◆ build RNA 5ʹ→3ʹ

COMPONENTS OF RNA Polymerase Holoenzyme

■ 2 αββ’ – core enzyme
■ (2 αββ’) δ- RNA Polymerase holoenzyme

 Transcription in Prokaryotes
■ Promoter sequences
enzyme
subunit RNA polymerase
read DNA 3ʹ→5ʹ
bacterial DNA
Promoter

TTGACA TATAAT
–35 sequence –10 sequence

RNA polymerase
molecules bound to
bacterial DNA

RNA polymerase
strong vs. weak promoters


INITIATION OF TRANSCRIPTION
 Transcription in Prokaryotes
■ Elongation
◆ RNA polymerase copies
DNA as it unwinds
■ ~20 base pairs at a time
300-500 bases in gene
■ builds RNA 5ʹ→3ʹ

Simple proofreading
■ 1 error/105 bases
■ make many mRNAs reads DNA 3ʹ→5ʹ

■ mRNA has short life

■ not worth editing!

2. ELONGATION
Transcription in Prokaryotes
■ Termination
◆ RNA polymerase stops at termination
sequence

 Termination of Transcription
■ Rho-independent termination-

◆ palindromic GC-rich region (hairpin loop) followed by a

stretch of AAAAAAAAA in the DNA being transcribed

 The G-C Hairpin

RNA GC
hairpin turn


❑ Rho-dependent termination

Rho- a hexamer which acts as a RNA-DNA helicase

GC region slows down RNA pol and Rho factor catches up and
dissociates RNA from transcription bubble

Rho
facto
r

Transcription in Eukaryotes

Transcription

RNA Processing

Translation

Protein
 Prokaryote vs. Eukaryote genes
■ Prokaryotes ■ Eukaryotes
◆ DNA in cytoplasm ◆ DNA in nucleus
◆ circular ◆ linear
chromosome chromosomes
◆ naked DNA ◆ DNA wound on
histone proteins
◆ no introns ◆ introns vs. exons

intron = noncoding (inbetween) sequence

eukaryotic
DNA
exon = coding (expressed) sequence

Transcription in Eukaryotes

■ 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 in Eukaryotes

■ Initiation complex
◆ transcription factors bind
to promoter region
upstream of gene
■ suite of proteins which bind
to DNA
turn on or off transcription
■ TATA box binding site
recognition site for
transcription factors
◆ transcription factors
trigger the binding of RNA
polymerase to DNA

 Part 2:
Post-transcriptional processing

■ Primary transcript (pre-mRNA)

◆ eukaryotic mRNA needs work after transcription

■ mRNA processing (making mature mRNA)

◆ mRNA splicing = edit out introns

◆ protect mRNA from enzymes in cytoplasm

■ add 5ʹ cap
■ add polyA tail
A t ail
poly- 3'
3' A A
AAA s
' c ap mRNA 0 A’
50-2
5
5 P
5' G PP



 Eukaryotic mRNA before

and after Processing

intron = noncoding (inbetween) sequence

~10,000 bases
yotic DNA
exon = coding (expressed) sequence
pre-mRNA
ry mRNA
ranscript
mature ~1,000 bases
mRNA spliced mRNA
transcri
pt

 RNA PROCESSING
1. Capping- addition of a methylated G nucleotide to the 5’ end of the
transcript
■ occurs right after about 30 nucleotides of RNA have been synthesized
■ protects growing RNA transcript from degradation
■ also for ribosome recognition during translation

CAPPING
 2. Tailing

- Addition of 100-200 residues of Adenylic acid or poly A’s to the 3’ end even
before termination of transcription has completed
-Due to enzyme Poly A Polymerase
-Sequence AAUAA located at 10-30 nucleotides upstream from site of cleavage
signals poly A polymerase to cleave a portion of the transcript before the poly A
tail is synthesized













 TAILING

1. aids in export of mature mRNA from nucleus; for RNA stability

2. prevents degradation from 3’ end
3. serves as recognition signal for ribosome

 3. Splicing

-introns are cut out of immature RNA transcripts

- mature transcripts code for proteins
- splicing signals tell spliceosomes where to ‘cut and paste’
exons
- introns have consensus sequences on their 5’ and 3’ ends
which are recognized by spliceosomes





















Alternative Splicing
 Splicing must be accurate
■ No room for mistakes!
◆ splicing must be exactly accurate
◆ a single base added or lost throws off the
reading frame

AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|UCC|GAU|AAG|GGC|CAU
Met|Arg|Ser|Asp|Lys|Gly|His
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
Met|Arg|Val|Arg|STOP|

 Part 3:

Translation

from
nucleic acid language
to
amino acid language






COMPONENTS OF THE TRANSLATION MACHINERY
1. Ribosome

- protein factory composed of aggregates of RNA and protein 70S in prokaryotes and 80S in
eukaryotes

THE RIBOSOME
 Ribosomes
■ A site (aminoacyl-tRNA site)
◆ holds tRNA carrying next amino acid to
be added to chain
■ P site (peptidyl-tRNA site)
◆ holds tRNA carrying growing
polypeptide chain Met
■ E site (exit site)
◆ empty tRNA
leaves ribosome
from exit site 5' U A C
A U G
3'
E P A



2. messenger RNA (mRNA)





3. Transfer RNA – ( tRNA)

Unfolded folded
 Transfer RNA structure
■ “Clover leaf” structure
◆ anticodon on “clover leaf” end
◆ amino acid attached on 3ʹ end

Loading tRNA
■ Aminoacyl tRNA synthetase
◆ enzyme which bonds amino acid to tRNA
◆ bond requires energy
■ ATP → AMP
◆ energy stored in tRNA-amino acid bond
■ unstable
■ so it can release amino acid at ribosome easily

Trp C=O Trp C=O Trp

OH H 2O
OH O

C
=O
O
activating
enzyme

tRNATrp AC C
anticodon UGG mRNA
tryptophan attached
to tRNATrp tRNATrp binds to UGG
condon of mRNA

 4. amino acyl synthetase-

enzyme that binds the specific amino acid with the tRNA being the
corresponding anticodon

Step 1 : AA + ATP - AA-AMP + PPi

Step 2: AA-AMP + tRNA - AA-tRNA + AMP















5. protein factors- for initiation, elongation and termination of translation

6. Peptidyl transferase- catalyzes

peptide bond formation

 FEATURES OF THE GENETIC CODE

•Universal- almost all organisms follow the same codon
assignments
Exceptions:
organism codon cytoplasm mitochondria

all UGA termination trp

mammal AUA ile met
yeast CUA leu thr
fruit fly AGA arg ser

•Triplet- three bases code for one amino acid

• Non-overlapping

AUU ACU GAU GGU – mRNA

Ile thr asp gly- polypeptide

mRNA codes for proteins in triplets

DNA TACGCACATTTACGTACGCGG
codon
mRNA AUGCGUGUAAAUGCAUGCGCC
?
protein Met Arg Val Asn Ala Cys Ala
The code
■ Code for ALL life!
◆ strongest support for
a common origin for
all life
■ Code is redundant
◆ several codons for
each amino acid
◆ 3rd base “wobble”

Why is the
wobble good?
■ Start codon
◆ AUG
◆ methionine
■ Stop codons
◆ UGA, UAA, UAG

 FEATURES OF THE GENETIC CODE

• Redundant or Degenerate- several codons code for one
amino acid

CUU, CUC, CUA, CUG- code for leu

• Structurally similar amino acids are represented by related
codons

• CUU codes for leucine ; AUU codes for isoleucine

• stop or termination codons signal end of translation ( UAA,

UAG, UGA)

• in vivo protein synthesis needs AUG (or GUG) as start

codon

Translation
■ Codons
◆ blocks of 3
nucleotides
decoded into
the sequence
of amino acids



 Bacterial chromosome

Translation in
Transcription
Prokaryotes mRNA

Translation

protein

Cell
membrane

Cell wall

 Translation in Prokaryotes

■ Transcription & translation are simultaneous

in bacteria
◆ DNA is in
cytoplasm
◆ no mRNA
editing
◆ ribosomes
read mRNA
as it is being
transcribed










Translation: prokaryotes vs. eukaryotes
■ Differences between prokaryotes &
eukaryotes
◆ time & physical separation between
processes
■ takes eukaryote ~1 hour
from DNA to protein
◆ RNA processing



 Building a polypeptide
■ Initiation
◆ brings together mRNA, ribosome
subunits, initiator tRNA
■ Elongation
◆ adding amino acids based on
codon sequence
■ Termination
◆ end codon 3 2 1
Leu Val release
Ser factor
Met Met
Met Met Leu Leu Leu Ala
Trp
tRNA
C
A

U AC U A C G A C AA U A C GA C U A C G A C AA U
5' C U GA A U 5'
A U G CU G U 5' A U G C UG AAU 5'
AU G C U G 3'
mRNA A U G 3' 3' 3' A CC
U GG U A A
E P A 3'

 1. Initiation of translation (Prokaryotes)

■ reactions prior to formation of the first peptide bond

■ a very slow step

❖Prokaryotes-requires binding of rRNA to Shine-Dalgarno sequence

(AGGA) located upstream of mRNA
❖first amino acid is formylated methionine (fmet)

 Eukaryotic Initiation of Translation
• starts with AUG nearest to the 5’ terminus of the mRNA
molecule (Scanning hypothesis)
•first amino acid incorporated is not fmet

2. Elongation of Translation
• reactions from synthesis of the first peptide bond to the last amino acid
most rapid step

3. Termination of Translation
steps needed to release the completed polypeptide chain
involves the dissociation of the ribosome; a very slow step