⑭

enc
·

F
~ ChapterB
SARA ERCIYAS
PROKARYOTES
crick frameshift mutation-bacteriophage T4

polynucleotide phosphorylase
* DNA template not needed
* random order of ribonucleotide
added
* artificial synthesis of RNA templates

UVU- Phenylalanine
Nirenberg & Matthaei RNA homopolymers :

AAA -
lysine
CCC- Proline
template (folda
4G -
notfunctional
, other
Nirenberg & Matthael & Ochoa RNA heteropolymers : based on probability
approaches still needed

Nirenberg & Philip & Leder Triplet binding assay

synthetic RNA triplet
(radioactive( > in vivo Ribosome
- ,
tRNA anticodon

and
Radioactively charged tRNA , RNA triplet and ribosomes were incubated together
. It retains ribosomes because of
their
then applied to a nitrocellulose filter
had bound to the RNA
charged tRNA
larger size. If radioactivity retained -
Ribosene
triplet associated with the
* SpecificCoda assignment should be
made .

had been tested

an incorrect aa
Radioactivity retainedI
T
If
* what did specific assignments led to discover
?
* 1) The genetic code is degenerate (2 ad may be specified by1triplet)
* 2) The code is unambiguous (a codon specifies a single aa)

Har Gobind Khorana Repeating Copolymers

RNA molecules Repeated Short Sequences
#Chemically Synthesized long
w

tell which one

* Ucu & CUC
specify leucine& serine but can't

* Reaffirmed
identity of triplets
2 triplets were termination codons
- at least
* GAVA-GUAA
UAA
G w

* only tryptophan & methionine are encoded by single codons.

Crick's Wobble Hypothesis

A critical in correct tRNA
and B are more attracting the
C present a more flexible adherence

VAA, UAG, UGA

Nonsense mutation >
- Mutation causing
Genetic Code
Phage MS2 (SSRNA) infect E Coli
.
,
study confirms
,

changes cytoplasm may represent

an evolutionary
note: in codon recognition in

toward the number human mitochondria.

trend reducing of tRNAs needed in

ORFs
different reading frames overlapping
different initiation points
overlapping genes
:

ORF : open reading frame = a gene

have Policistronic mRNA

note : Unlike Eukaryotes ,
prokaryotes
?
~ E2)
Es & How
Cluster of genes that
commonthe functionally related Al Openons .
are functionally related .

enzymes
exy Lac opera (lacZ , lac Y, lacA)
,
RNA polymerase do not need a primer
,

n(NTP) m(NMP)n + n(PPi)

NTP : nucleoside triphosphate (substrate

monophosphate
NMP nucleotides/nucleoside
:
bands)
5' to 3' phosphodiester
Ilinked by the triphosphate
(formed by Energy
released by dealing
PP :
inorganic diphosphate
precursor) .

?
do the equation look like as transcription progresses
a what
* PPi
(NMP)n + NTPm(NMP) n+
+

2 L Copies ,
B, B' ,
w
enzyme
:
core
um
site
holoenzyme : +o the catalytic mechanism & active
provide
.
for transcription

formation of +1
helix locally -
Promoters 7o factor transcription start
bind to denatured site
5 Regio,
in
-35
10
upstream of
-

assures RNA
coding gene positioned
pol is

↓
correctly
specific to promoter

consensus Sequences
conserved sequences

expribnow
box --10 TATAAT
a
what happens ifDo
exn-35 TTGACA TRANSCRIPTION or severely
Ay NO ,

Reduce the initiation of gene expression

 functional groups
cis acting element # "next to" other
* Regulatory sequences
- 10 - 35 +1
ex , ,

factor *"across from

"
trans acting
*proteins that influence gene expression by binding cis-elements

exofactor

cause Variation in time of initiation :

Bacteria have strong I weak promoters. They a

1to 2 seconds
& 310 to 20 minutes

?
& Major O subunit in Eukaryotes are
of 70 KDa However other several factors
molecular weight
.

A 070 based on its

,
Conditions (heat/Starvatia).
environmental
under different
also present (032 ,
054 ,
00E) ,
called
initiation
which provides specificity to transcription
.

Eacha factor recognizes different promoter sequences ,

?
a what happens
once RNA pol has recognized I bound to the promoter
Strand
AyDNA is locally converted to an open structure exposing the template
. .

* The first rNTP (5' end of the

transcriptis inserted at start site
* RNA
polymerization proceeds in 5'to 3' direction (in terms of the nascent RNA) creating
a temporary 8-bp DNA/RNA duplex whose chains run antiparallel to one another.
* O subunit dissociates (after these initial ribonucleotides are added
Chain
*
elongation proceeds under the direction of the core enzyme .
(50 nucleotides/s at 37C in E
.
coli)

* RNA pol performs proofreading as it adds each ribonucleotides. If a mismatch, enzyme backs up S correct .

bacteria why?
* Enzyme encounters termination Sequence(s) These sequences .
are extremely important in ,

Because the end of a

gene o the upstream gene are close together
.

*
Newly formed transcript form a hairpin secondary structure, held by H bands .

& What are the 2 transcription termination types in bacteria ?

1 Intrinsic termination (80 % of transcripts in E . Coli)

2 Rho-dependent termination.
 & Rho-dependent termination. helicase
RNA
a compare intrinsic A large hexameric protein with
.
activity (breaks H-bonds)

Il I
Eno-dependent
Intrinsic termination 80 %
termination
20 %

Rho protein (p)

*
Relies on the and a
mechanism relies on Self complementary GC-Rich -
termination sequence transcribed into a
sequences in the RNA transcript ,

hairpin structure in the RNA . Is dependent

forming a stable GC-Rich hairpin termination
on gene lacking strong intrinsic
tract.
Followed by a Poly-U signals (eXGC rich hairpin or
Poly-u tract
and But site presence (rho utilization
site)

Requires ATP For Rho's helicase

Energy Does not require additional
requirement
energy , spontaneous process activity

to stall rut
Rho binds to site Smoves along
Hairpin causes RNA polymerase
Details the RNA towards 3'end. When RNA
Weak A-U base pairing in the poly-u

↓
tract leads to dissociation of RNA pol pol pauses at a hairpin Rho catches up,
,

Release.
& transcript
dissociates the RNA-DNA hybrid S
terminates transcription.
only 2 H-bands

from bacteria ?
& Notable differences of Eukaryote transcription
A nucleus. For translation, mRNA moves to cytoplasm.
I occurs in
RNA
2 SRNA polymerases RNAP I-large
highly regulated on a gene-by-gene basis
.

12 subunits,
RNAP II-mRNA
E
,

nucleoplasmic RNA tRNA

RNAP III-Smaller ,

chromatin Fibers to be uncoiled

3
Requires Compact
Initiation : chromatin
remodeling
4 Several TFs are required : bind promoter ,
recruit RNAPol
,
initiate transcription, enhancers &Silencers

5 Complex termination
S produce mature mRNA : addition of 5'cap S S' PolyA tail removalof introns
6 pre mRNAs are processed , .

what ?
& RNAPI activity depends
on

trans-acting transcription factors that bind tothese elements

elements of the gene &
Ay on cis-acting regulatory
.

TATA box (-30) analogous to TATA-10

Golden-Hogness box ,

RNA pol do not directly bind to TATA

Eukaryotic
-
>
.
? in bacteria but now,
& cis-acting elements
· core promoter right : next to gene , more physical/structural ,
TF bind, help RNAPII
correctly position.
·
proximal promoter element slightly : upstream ,
how much when help modulate and where
,
the gene is expressed (level of transcription). Lots of if binds here.

3
enhancers
transcription initiation
·

influence efficiency or rate of the

by RNAPII from the core
silencers promoter element
> with
·

looping come to close . We can even find

chromatin ,
proximity
in an intron.
an enhancer element
 air groups promoter binding Nex TFID
① General TF (GTFs) ,
mandatory for all RNAPII core

TATA box
.
directly binds
activators/repressors.
② Transcriptional

bacteria, there is no specific sequence that signals for the termination. In fact, RNAPII
Note : Unlike
well beyond what will eventually be S'end of Mature MRNA Once AAUAA
transcription
.

continues
> 10-35 nucleotides after
-

(polyadenylation Signal sequence)

.

is incorporated, cleavage happen , RNAPII gets destabilized, clamp opens

,

both DNA &RNA are released from the Please note the
enzyme. importance of Kornberg's findings.

a post transcriptional
modifications in
Eukaryotic RNA :

Darnell)
*
Al 5'7-methylguanosine (m G) cap addition (discovered by Aaron Shatkin & James
protect nuclease attack at 5' end facilitates
, transport of mature mRNA
the cap has unique 5'to5' triphosphate in addition to a CH G at position I of base .

J
splicing
↓
poly-A polymerase Catalyzes addition of
poly-A tail (AAUAA) to S'OM .
(except histone coding
mRNAs in eukaryotes)
poly-A binding protein prevents
3'
degradation by . nucleases.

A In some eukaryotic cells , poly A tail is added before splicing of all introns has

been completed
.

& Poly A fails are also found in Archaea & Bacteria mRNAs .

associated with mRNA degradation though!

② One of the first intron containing gene identified ?
Ay B-globin gene, studied by Philip Leder & Richard Flavell in mice & rabbits .

Q Another example ?
Al Ovalbumin gene of chicken

identify introns ?
&, How to

A, compare sequence of DNA /mRNA correlation with amino acids

-94 % of human
protein coding genes have introns (8 out of 17)
ex collagen have 51 introns .

have NO intron ?
& which genes
Ay histone-coding , interferon-coding (Signalling protein of the immune
system)

a why do introns exist ?

1 gene can encode for more than 1 protein through alternative splicing. New genes may evolve through
shuffling ,
If Contains MiRNA
regulates gene expression (small
noncoding RNA)
exon .

.
Can also
regulate transcription (if harbor
cis-regulatory elements such as enhancers /silencers)
.

In +RNA ,
intron is cleared (endonuclease) , exons are sticked
by ligase.
&
Y
No extra help needed : RNA >
Ribozyme self excision GroupI introns Where
= =
=

do
.

we see
group I introns ?
Al MRNA, tRNA , rRNA in bacteria , lower eukaryotes higher plants.
,

cofactor &
a Group II introns?
>
-
primary
transcript

Ay, in Mitochondria Schloroplast of

fungi,plants, protist l bacteria .

Occurs for mRNAs

SpliceosomeComplex
> 500 000 nucleotides

phosphodiester

comparent of Spliceosore?
band broken

a essential
newly formed
(snRNAs) that

[
S'OH S phophodiester Ay small nuclear RNAs
bad interact

form snRNPs (small nuclear

ribonucleoproteins) .

Rich in U
 deleae
splice
r splicecepta
or
a RNA editing , prior to translation
I

↓ ↓
substitution insertion/deletion
editing (used in editing (mitoch.
nuclear derived eukaryotic RNA of slime
RNA)-ApoB MRNA
mold) (guide
edit CeV change - Stop
direct it
coder (chloroplast & gRNA
mitochondrial
.

in
Mitochondrial RNA of parasite
RNA of
a

plants) (mitochondrial Trypanosoma)

RNA of slime
mold)