Genetika Prokariot

Oswald Avery dan kawan-kawan pada tahun 1940 menunjukkan bahwa materi hereditas adalah
ADN dan bukan protein. Kemudian pada tahun 1953 James Watson dan Francis Crick
mengusulkan bahwa molekul ADN terdiri dari dua benang yang berikatan satu sama lain melalui
ikatan hidrogen. Kedua benang itu berjalan melingkari satu dengan lainnya sehingga membentuk
heliks.
Dogma sentral yang diusulkan oleh Francis Crick mengatakan bahwa informasi yang terkandung
di dalam ADN dapat ditranskripsikan menjadi ARN dan informasi di dalam ARN dapat
ditranslasikan menjadi protein.
Semenjak dogma sentral diperkenalkan untuk pertama kali, telah dipelajari bahwa beberapa virus
memproduksi enzim yang dapat menstranskripsikan ARN menjadi ADN dan virus-virus tersebut
dapat mereplikasikan genom ARNnya.
Proses sintesis ADN dengan ARN sebagai template disebut transkripsi balik. Transkripsi adalah
proses pembentuk ARN dengan model (template) ADN dan dikatalisis oleh enzim ARN
polimerase. Sedangkan transkripsi balik adalah sintesis ADN menggunakan ARN sebagai
template dan dikatalisis oleh enzim transkriptase balik. Proses translasi dilakukan oleh ribosom;
dan replikasi ADN dilakukan dengan bantuan kompleks ADN polimerase.
George Beadle dan Edward Tatum berjasa karena mampu mengumpulkan data untuk mendukung
hipotesis satu gen mengontrol aktivitas satu polipeptida (one gene one enzyme hyphotese)
pada eukariot rendah.
Bakteri umumnya mengandung satu genom ADN sirkuler yang melekat pada membran selnya.
Genom bakteri bereplikasi dimulai dari satu tempat menuju kedua arah (bidirectional). Satu
genom dapat melakukan replikasi lengkap dalam kurang dari setengah jam. Sejumlah enzim dan
protein terlibat dalam replikasi ADN seperti ARN polimerase yang membuat ARN primer, ADN
ligase, ADN polimerase.
Replikasi ADN berlangsung secara semikonservatif. Pada bakteri, genom anak terpisah satu
sama lain oleh pertumbuhan membran sitoplasma di antara titik perlekatannya. Sel anak
terbentuk bila membran sel dan dinding sel melakukan invaginasi dan membentuk pembatas di
antara kromosom.
Prokariot seperti halnya eukariot mensintesis tiga tipe ARN yaitu ARN-d, ARN-t, dan ARN-r.
ARN ribosom merupakan komponen struktural dari ribosom. Pada bakteri ARN-r 5S dan 23S
terdapat pada sub unit ribosom 30S.
Terdapat 1 sampai 6 ARN-t untuk masing-masing asam amino dari 20 asam amino yang dikenal
pembentuk protein. Ribosom mengkatalisis pembentukan ikatan peptida di antara dua asam
amino yang berdekatan.
Sintesis protein terdiri atas 3 tahap, inisiasi, elongasi, dan terminasi. Pada bakteri, selama inisiasi
ribosom 70S yang merupakan pertautan dari subunit 30S dan 50S pada kodon start AUG pada
ARN-d. Selama elongasi asam amino membentuk ikatan kovalen secara berurutan mengikuti
gerakan ribosom sepanjang ARN-d. Terminasi sintesis potein terjadi jika ribosom sampai kepada
kodon nonsens (UAA, UGA, UAG). Peptida selanjutnya terlepas dari ikatan ARN-t dan ribosom
berdisosiasi menjadi sub unitnya.
Kode genetika adalah triplet, karena masing-masing kode terbentuk dari tiga nukleotida yang

disebut kodon. Kodon terdiri dari tiga dari empat kemungkinan nukleotida (A, U, C dan G). Ini
berarti akan terdapat 43 = 64 kodon. Keenam puluh empat kodon ini dikenal oleh ARN-t kecuali
tiga yang disebut kodon nonsens.
Marshall Nirenberg dan Gobin Khorana berjasa dalam hal menemukan kode genetika ini. Dan
kode genetika ini berlalu universal untuk semua organisme dari bakteri sampai manusia.
Dasar

Transcription (genetics)
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A micrograph of ongoing gene transcription of ribosomal RNA illustrating the growing primary
transcripts. "Begin" indicates the 3' end of the DNA template strand, where new RNA synthesis
begins; "end" indicates the 5' end, where the primary transcripts are almost complete.
Transcription is the synthesis of RNA under the direction of DNA. Both nucleic acid sequences
use the same language, and the information is simply transcribed, or copied, from one molecule
to the other. DNA sequence is enzymatically copied by RNA polymerase to produce a
complementary nucleotide RNA strand, called messenger RNA (mRNA), because it carries a
genetic message from the DNA to the protein-synthesizing machinery of the cell. One significant
difference between RNA and DNA sequence is the presence of U, or uracil in RNA instead of the
T, or thymine of DNA. In the case of protein-encoding DNA, transcription is the first step that
usually leads to the expression of the genes, by the production of the mRNA intermediate, which
is a faithful transcript of the gene's protein-building instruction. The stretch of DNA that is

transcribed into an RNA molecule is called a transcription unit. A transcription unit that is
translated into protein contains sequences that direct and regulate protein synthesis in addition to
coding the sequence that is translated into protein. The regulatory sequence that is before, or 5',
of the coding sequence is called 5' untranslated (5'UTR) sequence, and sequence found
following, or 3', of the coding sequence is called 3' untranslated (3'UTR) sequence. Transcription
has some proofreading mechanisms, but they are fewer and less effective than the controls for
copying DNA; therefore, transcription has a lower copying fidelity than DNA replication.[1]
As in DNA replication, transcription proceeds in the 5' 3' direction. Only one of the two DNA
strands is transcribed. This strand is called the template strand, because it provides the template
for ordering the sequence of nucleotides in an RNA transcript. The DNA template strand is read
3' 5' by RNA polymerase and the new RNA strand is synthesized in the 5' 3' direction. RNA
polymerase binds to the 5' end of a gene (promoter) on the DNA template strand and travels
toward the 3' end. Except for the fact that thymines in DNA are represented as uracils in RNA,
the newly synthesized RNA strand will have the same sequence as the coding (non-template)
strand of the DNA. For this reason, scientists usually refer to the DNA coding strand that has the
same sequence as the resulting RNA when referring to the directionality of genes on DNA, not
the template strand.
Transcription is divided into 3 stages: initiation, elongation and termination.

Contents
[hide]
1 Prokaryotic vs. eukaryotic transcription
2 Initiation
3 Elongation
4 Termination
5 Measuring and detecting transcription
6 Transcription factories
7 History
8 Reverse transcription
9 References
10 See also
11 Further reading
12 External links

[edit] Prokaryotic vs. eukaryotic transcription

Prokaryotic transcription occurs in the cytoplasm along side translation.
Eukaryotic transcription is primarily localized to the nucleus, where it is separated from the
cytoplasm by the nuclear membrane. The transcript is then transported into the cytoplasm
where translation occurs.

Another important difference is that eukaryotic DNA is wound around histones to form
nucleosomes and packaged as chromatin. Chromatin has a strong influence on the
accessibility of the DNA to transcription factors and the transcriptional machinery
including RNA polymerase.
In prokaryotes, mRNA is not modified. Eukaryotic mRNA is modified through RNA
splicing, 5' end capping, and the addition of a polyA tail.[2]

[edit] Initiation
Simple diagram of transcription initiation. RNAP = RNA polymerase
Unlike DNA replication, transcription does not need a primer to start. RNA polymerase simply
binds to the DNA and, along with other cofactors, unwinds the DNA to create an initiation
bubble so that the RNA polymerase has access to the single-stranded DNA template.
In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA.
The RNA polymerase is a core enzyme consisting of five subunits: 2 subunits, 1 subunit, 1 '
subunit, and 1 subunit. At the start of initiation, the core enzyme is associated with a sigma
factor (number 70) that aids in finding the appropriate -35 and -10 basepairs downstream of
promoter sequences.
Transcription initiation is far more complex in eukaryotes and archaea,[3] the main difference
being that eukaryotic polymerases do not recognize directly their core promoter sequences. In
eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA
polymerase and the initiation of transcription. Only after certain transcription factors are attached
to the promoter does the RNA polymerase bind to it. The completed assembly of transcription
factors and RNA polymerase bind to the promoter, called transcription initiation complex.
In prokaryotes RNA Polymerase bind the mRNA and then forms a "closed complex". This
complex is unwound to create the open complex which has melted DNA from -12 to +2.

[edit] Elongation
Simple diagram of transcription elongation
One strand of DNA, the template strand (or non-coding strand), is used as a template for RNA
synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses
base pairing complementarity with the DNA template to create an RNA copy. Although RNA
polymerase traverses the template strand from 3' 5', the coding (non-template) strand is
usually used as the reference point, so transcription is said to go from 5' 3'. This produces an
RNA molecule from 5' 3', an exact copy of the coding strand (except that thymines are
replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA
has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).
Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single
DNA template and multiple rounds of transcription (amplification of particular mRNA), so many

mRNA molecules can be produced from a single copy of a gene. This step also involves a
proofreading mechanism that can replace incorrectly incorporated bases.
Prokaryotic elongation starts with the "abortive initiation cycle". During this cycle RNA
Polymerase will synthesize mRNA fragments 2-12 nucleotides long. This continues to occur
until the factor rearranges, which results in the transcription elongation complex (which gives a
35 bp moving footprint). The factor is released before 80 nucleotides of mRNA are
synthesized.

[edit] Termination
Simple diagram of transcription termination
Bacteria use two different strategies for transcription termination: in Rho-independent
transcription termination, RNA transcription stops when the newly synthesized RNA molecule
forms a hairpin loop, followed by a run of Us, which makes it detach from the DNA template. In
the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the
interaction between the template and the mRNA, thus releasing the newly synthesized mRNA
from the elongation complex. Transcription termination in eukaryotes is less well understood. It
involves cleavage of the new transcript, followed by template-independent addition of As at its
new 3' end, in a process called polyadenylation.

[edit] Measuring and detecting transcription

Transcription can be measured and detected in a variety of ways:
Northern blot
RNase protection assay
RT-PCR
In vitro transcription
In situ hybridization
DNA microarrays

[edit] Transcription factories

Active transcription units are clustered in the nucleus, in discrete sites called transcription
factories. Such sites could be visualized after allowing engaged polymerases to extend their
transcripts in tagged precursors (Br-UTP or Br-U), and immuno-labeling the tagged nascent
RNA. Transcription factories can also be localized using fluorescence in situ hybridization, or
marked by antibodies directed against polymerases. There are ~10,000 factories in the
nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000
polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active
transcription units are associated with only one polymerase, each factory will be associated with

~8 different transcription units. These units might be associated through promoters and/or
enhancers, with loops forming a cloud around the factory.

[edit] History
A molecule which allows the genetic material to be realized as a protein was first hypothesized
by Jacob and Monod. RNA synthesis by RNA polymerase was established in vitro by several
laboratories by 1965; however, the RNA synthesized by these enzymes had properties that
suggested the existence of an additional factor needed to terminate transcription correctly.
In 1972, Walter Fiers was the first person that actually proofed the existence of the terminateenzyme.
Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies of the molecular
basis of eukaryotic transcription".[4]

[edit] Reverse transcription

Scheme of reverse transcription

Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA.
HIV has an RNA genome that is duplicated into DNA. The resulting DNA can be merged with
the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an
RNA template is called reverse transcriptase. In the case of HIV, reverse transcriptase is
responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. An
associated enzyme, ribonuclease H, digests the RNA strand, and reverse transcriptase synthesises
a complementary strand of DNA to form a double helix DNA structure. This cDNA is integrated
into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral
proteins which reassemble into new viral particles. Subsequently, the host cell undergoes
programmed cell death (apoptosis).
Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase.
Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase
carries an RNA template from which it synthesizes DNA repeating sequence, or "junk" DNA.
This repeated sequence of "junk" DNA is important because every time a linear chromosome is
duplicated, it is shortened in length. With "junk" DNA at the ends of chromosomes, the
shortening eliminates some repeated, or junk sequence, rather than the protein-encoding DNA
sequence that is further away from the chromosome ends. Telomerase is often activated in cancer
cells to enable cancer cells to duplicate their genomes without losing important protein-coding
DNA sequence. Activation of telomerase can be part of the process that allows cancer cells to
become immortal.

[edit] References
1. ^ Berg J, Tymoczko JL, Stryer L (2006). Biochemistry, 6th ed., San Francisco: W. H.
Freeman. ISBN 0716787245.
2. ^ Robert J. Brooker Genetics: analysis and principles. 2nd edition. (New York: McGrawHill 2005) Chapter 12 "Gene transcription and RNA modification" pp. 318-325.
3. ^ Mohamed Ouhammouch, Robert E. Dewhurst, Winfried Hausner, Michael Thomm, and

E. Peter Geiduschek (2003). "Activation of archaeal transcription by recruitment of the

TATA-binding protein". Proceedings of the National Academy of Sciences of the United
States of America 100 (9).
4. ^ Chemistry 2006. Nobel Foundation. Retrieved on 2007-03-29.

Reverse transcription
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Reverse transcription is the process of making a double stranded DNA (deoxyribonucleic acid)
molecule from a single stranded RNA (ribonucleic acid) template. It is called reverse
transcription as it acts in the opposite or reverse direction to transcription. This idea was very
unpopular at first as it contradicted the Central Dogma of Molecular Biology which states DNA
is transcribed into RNA which is translated into proteins. However, in 1970 when the scientists
Howard Temin and David Baltimore both independently discovered the enzyme responsible for
reverse transcription, named reverse transcriptase, the possibility that genetic information could
be passed on in this manner was finally accepted.

[edit] Reverse transcription in Class VI viruses

Class VI viruses ssRNA-RT, also called the retroviruses are RNA reverse transcribing viruses
with a DNA intermediate. Their genomes consist of two molecules of positive sense single
stranded RNA with a 5' cap and 3' polyadenylated tail. Examples of retroviruses include Human
Immunodeficiency Virus (HIV) and Human T-Lymphotropic virus (HTLV). Once the viruses have
entered the cell and been uncoated the genome is reverse transcribed into double stranded DNA
which can be incorporated into the host cell and subsequently expressed. Reverse transcription
by the enzyme reverse transcriptase occurs in a series of steps:
1. A specific cellular tRNA acts as a primer and hybridizes to a complementary part of the
virus genome called the primer binding site or PBS
2. Complementary DNA then binds to the U5 (non-coding region) and R region (a direct
repeat found at both ends of the RNA molecule) of the viral RNA
3. A domain on the reverse transcriptase enzyme called RNAse H degrades the 5 end of the
RNA which removes the U5 and R region
4. The primer then jumps to the 3 end of the viral genome and the newly synthesised
DNA strands hybridizes to the complementary R region on the RNA
5. The first strand of complementary DNA (cDNA) is extended and the majority of viral
RNA is degraded by RNAse H
6. Once the strand is completed, second strand synthesis is initiated from the viral RNA
7. There is then another jump where the PBS from the second strand hybridizes with the
complementary PBS on the first strand
8. Both strands are extended further and can be incorporated into the hosts genome by the
enzyme integrase

Mechanism of Reverse Transcription

After the RNA retrovirus enters a host cell, its genomic RNA will be transcribed into a
double stranded DNA and then integrated into the host DNA. The RNA to DNA transcription is
called reverse transcription.

Figure 4-J-1. Mechanism of reverse transcription. The entire process is catalyzed by reverse
transcriptase which has both DNA polymerase and RNase H activities.
1. A retrovirus-specific cellular tRNA hybridizes with a complementary region called the
primer-binding site (PBS).
2. A DNA segment is extended from tRNA based on the sequence of the retroviral genomic
RNA.
3. The viral R and U5 sequences are removed by RNase H.
4. First jump: DNA hybridizes with the remaining R sequence at the 3' end.
5. A DNA strand is extended from the 3' end.
6. Most viral RNA is removed by RNase H.
7. A second DNA strand is extended from the viral RNA.
8. Both tRNA and the remaining viral RNA are removed by RNase H.
9. Second jump: The PBS region of the second strand hybridizes with the PBS region of the
first strand.
10. Extension on both DNA strands. LTR stands for "long terminal repeat".