TRANSKRIPSI

SINTESIS RNA : mRNA, tRNA, rRNA

Study objectives
You will need to know ALL the concepts and details in this lecture.
1. What are the three main types of RNA and what are their functions?
2. Understand how the following terms apply to RNA structure: phosphodiester
bonds, 5' and 3 ends, nucleosides, complementary base pairing, stem loops.
3. Compare and contrast DNA and RNA structure.
4. What is a gene? What is gene expression? *Understand transcription,
translation, and RNA processing in both prokaryotes and eukaryotes.
5. Define operons and polycistronic messages. How do they function in
prokaryotic gene expression?
6. *Compare and contrast the features of prokaryotic and eukaryotic gene
expression. Do eukaryotes have operons? What are exons, introns, primary
transcripts, capping, tailing, and splicing. What is the 5' cap (methylguanosine
cap)? How and when is the poly-A tail added to the transcript? Where does
eukaryotic RNA processing occur?
7. Understand the structure and function of promoters and terminators in
transcription. Contrast intrinsic terminators and rho-dependent terminators.
8. Know the subunit structure of bacterial RNA polymerase and the sigma cycle.
DOGMA SENTRAL
Prokaryotic transcription
A. overview
B. transcribed regions
C. RNA polymerase
D. promoters
E. terminators
F. sigma factor
TB
Overview of prokaryotic transcription
RNA synthesis from a DNA template
typical
gene dsDNA

RNA polymerase

primary transcript complementary to

one strand of the coding region
TB
Defined regions are transcribed

upstream transcribed downstream

region region region

gene dsDNA

promoter transcription termination

start site site
(RNA polymerase
binding site)
TB
A. Functions of the major RNAs
1. messenger RNAs (mRNA) contain
genetic information to encode a protein
phe
2. transfer RNAs (tRNA) act as adapters
between the mRNA nucleotide code and
amino acids during protein synthesis
3. ribosomal RNAs (rRNA) are structural and
catalytic component of ribosomes
2. The phosphodiester bonds
of RNA are analogous to those
of DNA.

3. The 5' and 3' ends

of RNA are analogous to
those of DNA.
TB
 P-O-C ring numbering
O
5' end system for ribose
5'
OH -C O
phosphodiester
4’ 1’
bond O O
P 3’ 2’
O- O
C
O

3’ end HO OH RNA TB
Complementary base pairing

CCCTTTGGGAAA DNA
GGGAAACCCUUU RNA
hydrogen
bonding
GGGAAACCCUUU RNA
CCCUUUGGGAAA RNA
TB
Gene numbers
approximate
group gene number

viruses 4-200
prokaryotes 500-12,000
eukaryotes 5,000-125,000

TB
Any given species has a unique set
of genes that confers a unique set
of properties.
Proteins and RNAs determine all of the
characteristics of organisms and cells.
Example: Escherichia coli has 4405 genes
~117 encode RNAs (tRNA, rRNA)
~4288 encode proteins TB
Gene expression in prokaryotes
1. Expression of single genes
Ex.1: a single gene that encodes a protein
1 gene
transcription
1 mRNA
translation
1 polypeptide
TB
Ex. 2: a single gene that encodes one
rRNA or tRNA

1 gene
transcription
1 RNA
RNA processing
1 tRNA etc.
degraded TB
2. Expression of operons
operon A B C
DNA
two or more genes
transcribed together transcription

polycistronic message
polycistronic
a single RNA molecule that mRNA

represents more than one gene

TB
b. Operons can encode several
rRNA molecules.
1 operon

1 polycistronic
processing RNA

rRNA
rRNA
degraded
2 or more rRNAsTB
Important points
The details of organization,
processing and degradation are
different for different RNAs.
Most prokaryotes use operons.
Operons are used to coordinate
gene expression and often contain
genes of related function.
TB
 GENA
 Promoter
 Operator
 Lokus penempelan aktivator
 Struktural gena : mRNA
 Terminator
Polisistronik
Monosistronik
 TRANSKRIPSI = SINTESIS RNA
(mRNA, tRNA, rRNA)
 :
 Polimerase RNA
 ATP, CTP, GTP, UTP

 Urutan pada DNA:

 Promoter
 Operator
 Situs penempelen aktivator
 Terminator
 Gambaran dasar sintesis RNA
 Prekursor : ATP CTP GTP UTP
 Polimerase RNA
 Reaksi polimerisasi
 Urutan RNA ditentukan DNA
 Salah satu untai DNA sebagai catakan
 Untai RNA tumbuh dari 3’
 Polimerase mulai sintesis tanpa primer
C. RNA polymerase is the enzyme that
synthesizes RNA from a DNA template.
RNA polymerase
gene,or
operon

DNA template
complementary RNA
TB
+
+ completed
transcript
TB
1. Subunit structure of bacterial
RNA polymerase

core enzyme holoenzyme

' '

The holoenzyme includes one

of several sigma factors. TB
 The assembly pathway of the core enzyme

    ’ = core enzyme

I II CORE ENZYME

Sequence-independent,
nonspecific transcription
 ’ initiation

70
+ 3 54 SIGMA SUBUNIT
interchangeable,
vegetative heat shock promoter recognition
nitrogen starvation
(principal ) (for emergencies) (for emergencies)
 I II
RNAP HOLOENZYME -70
 ’ Promoter-specific
transcription initiation
70

In the Holoenzyme:

· ' binds DNA

·  binds NTPs
·  and  ' together make up the active site
·  subunits appear to be essential for assembly and
for activation of enzyme by regulatory proteins. They
also bind DNA.
·  recognizes promoter sequences on DNA
Binding of polymerase to Template
DNA

 Polymerase binds nonspecifically to DNA with

low affinity and migrates, looking for promoter.
 Sigma subunit recognizes promoter sequence.
 RNA polymerase holoenzyme and promoter
form "closed promoter complex" (DNA not
unwound).
 Polymerase unwinds about 12 base pairs to
form "open promoter complex“.
The sigma factor cycle
Sigma factors ( ) are a subunit
of RNA polymerase.

Sigma factors are needed for promoter

binding, but after transcription starts they
dissociate.

TB
INISIASI
 RNA pol holoenzyme (core + sigma)

sigma
factor core enzyme

sigma factor
RNA (~10 nucleotides)
TB
 termination
RNA
+
core enzyme

sigma

holoenzyme TB
Upstream region of the lactose operon
TAATGTGAGTTAGCTCACTCATTA
-35 region
GGCACCCCAGGCTTGACATTTATG
-10 region (Pribnow)
CTTCCGGCTCGTATGTTGTGTGGA
Transcription

AATTGTGAGCGGATAACAATTTCA
start site

Shine-dalgarno (RBS)
CACAGGAAAGAGCTATGACC...
Translation start site
Promoters
Sites on DNA where RNA
polymerase binds to start transcription
upstream transcribed downstream
region region region
gene dsDNA

transcription termination
promoter
start site site
TB
1. Typical bacterial 70 promoter

TTGACA TATAAT
AACTGT ATATTA
TTGACA = TATAAT =
-35 consensus -10 consensus
sequence sequence

*also called Pribnow box;

~ 10 bases before start
site of transcription TB
A more common way to draw a promoter

5' 3'
TTGACA TTAACT
-35 -10
Note:
The - 10 and -35 sequences can vary somewhat.

TB
SUSUNAN PROMOTER DAN OPERATOR OPERON lac
FOOTPRINTING
PROMOTER PROKARIOT
PROMOTER PROKARIOT & EUKARIOT
Konsensus
TATAAT
Konsensus
TTG- - -T
PERPANJANGAN TRANSKRIPSI
Transcriptional terminators
DNA region that mediates the
termination of transcription.
termination
site
gene dsDNA

region where
terminators are
usually found
TB
1. Intrinsic terminator
DNA encoding an RNA that forms
a stem loop followed by a run of "U"s
that is used for transcriptional termination.

UUUU
RNA
3' end of RNA
TB
Intrinsic terminator function

The RNA stem loop

binds to RNA pol and
causes termination

Important fact: Intrinsic terminators must be

transcribed in order to function. TB
2. Rho-dependent terminator
A DNA site where RNA polymerase
pauses and transcription
is terminated by Rho protein

TB
Rho protein binds RNA then moves
along RNA until it contacts RNA pol and
terminates transcription

RNA pol
pauses at
Rho
termination site
Rho protein TB
 Heksamer Rho
 Rho menempel RNA bergerak pada untai
tunggal mRNA dengan menghidrolisa ATP
 Ketika Rho mencapai polimerase,
memecah hibrid RNA-DNA
PROMOTER JENIS LAIN
 Mechanisms of antibacterial action

RNA polymerase DNA topoisomerases

cell wall rifampicin
quinolones,
-lactams novobiocin
vancomycin
bacitracin
DNA

mRNA
cell membrane 50S 50S 50S
polymyxins THF 30S
30S 30S

ribosomes
DHF

folate synthesis
sulphonamides
trimethoprim protein synthesis (30S) protein synthesis (50S)
pABA tetracyclines macrolides
aminoglycosides lincosamides
fusidic acid and others chloramphenicol
oxazolidinones
 Mutasi M.tuberculosis resistensi
obat

Hajj et al, 2001, Detection of rifampin resistance in Mtuberculosis in a single tube with
Molecular beacons, J.Clin.Microbiol., 39: 4131-4137
 Antinomycin D
 Interkalasi pada DNA – mencegah DNA
sebagai cetakan
 Pada dosis rendah menghabat transkripsi
tanpa efek pada replikasi
 Pada dosis tinggi menghambat replikasi
 tRNA assists in translation
 Transfer RNA takes a specific amino acid to the
ribosome
 Single stranded but has areas where bases hydrogen
bond to each other
 Cloverleaf shape
 3’ end picks up the amino acid
 Which amino acid is determined by 3 bases opposite the
amino acid called an anti-codon
 Anti-codons pair with complementary codons in mRNA at
a ribosome
(a) Structure of 16S rRNA
(b) Structure of 23 S rRNA
 STABILITAS RNA
 mRNA t ½ 2-3 menit,
 cara pengendalian
 Microarrays
 Traditionally, one gene and protein and
structure were studied.
 Insufficient to study cell processes and
life sciences.
 Cluster different genes and proteins to
study their relationships, functional
hierarchies.
 Microarray analysis
Microarrays are mRNAs
Spotted at high density onto
glass chips. Expression of
thousands of genes over
hundreds of cell states is
measured.

Identifying coregulated
genes is not so simple.
Many physicists work
in this field-- one recent
publication:
Super-paramagnetic clustering
of data , Eytan Domany,
Physica A 263, 158 (1999)
Microarray Data