Kafrelsheikh University

Faculty of Agriculture
Genetics Department

The Principals of Genetic

Lectures
Dr. Abdel-Hamid Ali
Prof. Of Cyto-biochemical Genetics &
Plant Biotechnology
Transcription

( )88
 The Central Dogma

Transcription Translation
1 2
DNA RNA PROTEIN
replication

4
 Gene expression
Gene expression
DNA
RNA RNA
.
Central Dogma
Proposed by Francis
Crick 1958
Transcription and translation in
eukaryotic cells are separated in
space and time.

Extensive processing of primary

RNA transcripts in eukaryotic cells.
 What is a gene?
Theentire nucleic acid sequence that is
necessary for the synthesis of a functional
polypeptide or RNA molecule.

.RNA
 DNA
Gene
Gene
Expression
Transcription

RNA (messenger RNA)

Translation

Protein
(sequence of
amino acids)

Functioning of proteins within living

cells influences an organisms traits.
LE 17-3-2

GENE

DNA
TRANSCRIPTION

mRNA
Ribosome
Prokaryotic cell

Polypeptide

Prokaryotic cell
 II. Transcription: RNA from DNA
What is the enzyme that can direct RNA synthesis?
RNA
RNA polymerase
RNA pol.
- ( NTPs ) ( ribose (
-
DNA
n(NTP) (NMP)n + n (Ppi)
enzyme
RNA pol.

RNA polymerase well characterized in E. coli - 6 subunits

 Making Sense of the Strands

DNA coding strand = Sense Strand

DNA template strand = Antisense Strand
mRNA formed = Sense Strand

Coding or Partner strand

5 3

mRNA 5 3

3 5
Template strand
Components of Prokaryotic Transcription

RNA pol.

NO primer required

5 to 3

50 nt/second
Prokaryotic Promoter Lies Just Upstream
(5) of Transcribed Region
Two Consensus sequences

-35 Region -10 TATA Box

Effect of mutations here?

 transcription
RNA
DNA RNA
DNA

RNA
polymerase
:
( )1 Initiation :
( )2 Elongation :
( )3 Termination
 TRANSCRIPTION

ACGATACCCTGACGAGCGTTAGCTATCG
UGCUAUGGGACU
 RNA polymerase
Prokaryotes
RNA polymerase
480.000

1 , 1, 2, ,
( )hole enzyme

) DNA (binding site promotor
 DNA Sequences Important to
Transcription in Prokaryotes
Promoter
Pribnow Box (also called the -10 element)
TATAAT
-35 element - TTGACA
 Holoenzyme

The holoenzyme of RNA-pol in E.coli

consists of 5 different sHoloenzyme
ubunits: 2 .

holoenzyme
core enzyme



Holoenzyme
 RNA-pol of E. Coli

subunit MW function
Determine the DNA to be
36512
transcribed

150618 Catalyze polymerization

155613 Bind & open DNA template

Recognize the promoter
70263
for synthesis initiation
RNAP - RNA pol.


RNAP
RNAP

.

' '
.
.

.
 Initiation :
RNA polymerase
DNA

RNA DNA

RNA Pol
. DNA
Promoter
Start site Start point

RNA -5
)(ribonucleoside 5'-triphpsphates
RNA
) . )triphosphate -'3
OH
'5 . :
 Transcription initiation by prokaryotic RNA polymerase
Holoenzyme sliding and scanning
Promoter
-35 -10

Closed complex

Sigma separates
rNTPs from the core
PPi once a few
phosphodiester
Core enzyme bonds are formed
Open complex; initiation


5pppA
mRNA

-1
)(3-OH

RNA )(DNA, RNA
.
-2 DNA

- RNA
DNA
.
 Elongation
RNA DNA, RNA

-3 RNA
DNA .
RNA

DNA
base-pair .
DNA C T G A
DNA G A C U
RNA .

-4
DNA


phosphodiester bond
DNA


.

'3 -5 RNA '5
OH -'3
RNA RNA
DNA .
-6 RNA polymaerase
DNA polymerase
RNA polymerase
primer.
 Elongation
Polymerase is accurate - only about 1 error in
10,000 bases
Elongation rate is 20-50 bases per second -
slower in G/C-rich regions (why??) and faster
elsewhere.


.
Phosphodiester
RNA
DNA RNA DNA
RNA DNA
.Terminator
Two termination models in E. coli
Two termination models in E. coli involve palindromes
1- core enzyme
In vivo
)rho- independent terminators) rho factor
.Simple terminator
** -:
hair pin ))terminators
u ( )6-4 RNA .run of u

2- rho factor
rho factor .
rho in vivo.
 FIGURE 27: Bacterial
termination occurs at a
discrete site
 19.15 Bacterial RNA Polymerase
Terminates at Discrete Sites
Intrinsic termination requires
recognition of a terminator
sequence in DNA that codes for
a hairpin structure in the RNA
product.
The signals for termination lie
mostly within sequences already
transcribed by RNA polymerase,
and thus termination relies on
scrutiny of the template and/or
the RNA product that the
polymerase is transcribing. FIGURE 28: An intrinsic
terminator has two features
 rho factor
rho factor

RNA 3
rho-dependent
termination rho-dependent term

hairpin G C

hairpin U A-
T.
Rho factor-dependent
 FIGURE 29: Rho
terminates transcription

Transcription in eukaryotes

RNA
:

-1
RNA polymerase
RNA RNA polymerase
I, II, III
 DNA Genotype

Transcription

RNA
Nucleus

Cytoplasm
Translation

Phenotype

Protein

Flow of genetic information in a eukaryotic cell

 -1
RNA polymerase

-2 '5 '3
cap '5
mRNA. '3 (
200) polyadenylic acid
) poly (A mRNA.
RNAP II transcripts, the pre-mRNAs, are
processed before export & translation

1-Addition of 5 7methyl-Gppp cap.

2- Addition of 3 poly-adenylated tails.
3-Splicing remove introns.
 Function of poly(A) tail
Increased mRNA stability
Increased translational
efficiency
Splicing of last intron

49
 The processing steps are:
Addition of a 5 7-methyl guanosine cap
(capping).

Addition of a poly-A tail at the 3 end

(polyadenylation)

RNA splicing to remove intervening sequences

(remove introns).
 Eukaryotic pre-mRNA Splicing
In eukaryotes, the primary transcript must be
modified by:
addition of a 5 cap
addition of a 3 poly-A tail
removal of non-coding sequences
(introns)

51
 -3 intervening sequences

introns mRNA
mRNA .
splicing processing
DNA GENE

transcription

messenger RNA
(mRNA)
translation

protein
 exon 1 intron exon 2
DNA GE NE
transcription

intron
precursor-mRNA
(pre-mRNA)
The intron is also present in the RNA copy of
the gene and must be removed by a process call
RNA splicing
 exon 1 exon 2
intron
DNA GE NE

transcription
intron
pre-mRNA

RNA splicing

mRNA

translation

protein
 exon intron exon
pre-mRNA M7G AAAAAAA200
cap poly(A) tail
RNA splicing

mRNA M7G AAAAAAA200

nucleus
transport

cytoplasm
M7G AAAAAAA200

ribosomes
protein
 intron 1 intron 2 intron 3 intron 4
1 2 3 4 5

1 2 3 4 5

Usually all introns must be removed before the

mRNA can be translated to produce protein
 Heart muscle 1 2 3 5

1 2 3 4 5

Uterine muscle 1 3 4 5

Thus one gene can encode more than one protein. The proteins are
similar but not identical and may have distinct properties. This is
important in complex organisms
 intron branchpoint

pre-mRNA A

Step 1

intermediates

Step 2

spliced mRNA
 spliceosome
(~100 proteins + 5 small RNAs)

pre-mRNA spliced mRNA

In eukaryotes mRNA must be processed
and transported out of nucleus for
translation

iGenetics, 1st ed. Russell

 Splice Sites
Conserved splice sites are shared by both
the exon and the intron.
Different signals on the donor site (3) and
on the acceptor site (5).

62
Gene Structure
Thegenes are made up of DNA
sequences.
-Promoters
-Exons
-Introns

-4 mRNA
monocistronic

Biochemistry 2/e - Garrett & Grisham

In Prokaryotic Polycistronic

coding sequence

Copyright 1999 by Harcourt Brace & Company

 In Eukaryotic monocistronic

promoter coding sequence

Prokaryotic vs eukaryotic mRNA molecules
 -5
-5 mRNA
.

 mRNA eukaryotes
Prokaryotes reticulocytes
mRNA
.
mRNA
mRNAs .silk fiberion, ovalbumin, globin

Dr/ Abdel -Hamid Ali
Exons and introns
Genes comprise only about 2% of the human genome;
the rest consists of non-coding regions, whose functions
may include providing chromosomal structural
integrity and regulating when, where, and in what
quantity proteins are made (regulatory regions).

The terms exon and intron refer to coding

(translated into a protein) and non-coding DNA,
respectively.
Structure of a typical prokaryotic mRNA molecule
The Protein Players - RNA polymerases, transcription factors, initiation
factors, enhancers, repressors
 Protein Synthesis: Translation
Translation is the process of decoding a mRNA
molecule into a polypeptide chain or protein.
mRNA
.
Each combination of 3 nucleotides on mRNA is called a
codon or three-letter code word.
Each codon specifies a particular amino acid that is to be
placed in the polypeptide chain (protein).
mRNA 3
.
.) (
Protein Synthesis: Translation
 OH
A Codon
NH2
HO P O

O
N

N
N Adenine
CH2 N
O

O H
O
HO P O

O
N
NH Guanine
N N NH2
CH2
O

HO
O

P
H
NH2
Arginine
O

O
N
N
Adenine
N N
CH2
O

OH H
 The Genetic Code
Marshall Nirenberg identified the codons
that specify each amino acid.
RNA molecules of only 1 nucleotide and of
specific 3-base sequences were used to
determine the amino acid encoded by
each codon.
The amino acids encoded by all 64 possible
codons were determined.

78
79
 The Genetic Code
stop codons: 3 codons (UUA, UGA, UAG)
in the genetic code used to terminate
translation
start codon: the codon (AUG) used to
signify the start of translation
The remainder of the code is degenerate
meaning that some amino acids are
specified by more than one codon.

80
 Transfer RNA
anticodon- 3 to 5 sequence that matches
the complementary 5 to 3sequence
(codon) on the mRNA
Acceptor arm - Amino acid code on 3 end
T and D loops provide structure for
interface with aminoacyl-tRNA synthetase

?
)Transfer RNA (tRNA

80 - 75








tRNA

Transfer RNA Bound to

one amino acid on one
end
Anticodon on the other
end complements mRNA
codon.
tRNA
'3
CCA


anticodon


mRNA
 Processing of pre-tRNAs

RNase P
cleavage
site
Protein Synthesis: Translation



rRNA




)S :S Svedberg unit
))sedimentation

Polysomes

Ribosomes

2 subunits, separate in cytoplasm

until they join to begin translation
Large
Small
Contain 3 binding sites
E (exit).
P (peptidyl)
A (aminoacyl)
 ( of ) Assembly
ribosomes in Eukaryotes

-3 .. .
)Ribosomal RNA (rRNA

45S

3
rRNA 28 S, 5.8 S

60S
5.8S
.40S
5S rRNA

rRNA

DNA
RNA RNA
.

rRNA
 The ribosomal RNA gene

Transcription by RNA Polymerases I

Large subunit does???? Small subunit does ?????


Thanks