replication expession

8/11/2019 Replication Expession

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DNA REPLICATION and

DNA EXPRESSION


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DNA Structure


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DNA Molecules

Consist of: Ribose

Phosphate

Nucleotide base

Organization: Consist of 2 polymers of ribose & phosphate that

connect each other by nucleotide base pairs

The 2 polymers of ribose & phosphate runs in

opposite dirrection Nucleotide base pairs are

AdenineThymine

Guanine - Cytocyne


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Forms of DNA

DNA exists in many possible conformations. only A-DNA, B-DNA, and Z-DNAhave been

observed in cells.

conformation depends on:

the DNA sequence

the amount and direction of supercoiling

chemical modifications of the bases

solution conditions (eg concentration of

metal ions, salt concentration and level of

hydration).


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ADNA B-DNA Z-DNA
http://upload.wikimedia.org/wikipedia/commons/1/13/A-B-Z-DNA_Side_View_Transparent.png


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B-DNA

The common form of DNA

Has 10 base pairs per turn; One turn spans 3.4 nm

A-DNA

When higher salt concentrations or alcohol added,

the DNA structure may change to A form Has 11 bases per turn; One turns spans 2.3 nm

Z-DNA

The strands turn about the helical axis in a left-handed spiral.

Has 12 bases per turn; One turn spans 4.6 nm


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DNA Replication in Bacteria

In general, DNA is replicated by: uncoiling of the helix

strand separation by breaking of the

hydrogen bonds between the complementary

strands

synthesis of two new strands by

complementary base pairing

Replication begins at a specific site in the DNA

called the origin of replication(ori)

DNA replication is bidirectionalfrom ori


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Replication Fork


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To begin DNA replication, unwinding enzymescalled DNA helicases cause the two parent DNAstrands to unwind and separate from one anotherat the origin of replication to form two "Y"-shapedreplication forks

Helix destabilizing proteinsbind to the single-stranded regions so the two strands do not rejoin

Enzymes called topoisimerasesproduce breaks inthe DNA and then rejoin them in order to relievethe stress in the helical molecule during replication.

New complementary strands are produced by thehydrogen bonding of free DNA nucleotides withthose on each parent strand


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The new molecule run on 53 direction

The First molecule on complementary strand are RNA

(called RNA primer) )because Primase can only

synthesis new molecules without preexisting

molecule.

After RNA primer, DNA polymerase begin a new DNA

chain continuing RNA primer

The RNA primer is later degraded and filled in with

DNA

The gap between new DNA and another joining by

Ligase


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Leading vs Lagging strand

The DNA consist 2 strand

The new DNA emerged from each DNAparental strand

Each new DNA run in opposite direction(bidirectional)

There are 2 type new DNA strand (leading andlagging strand) in each DNA parental strand

Leading strand means new DNA synthesis oneRNA primer and long growing DNA

Lagging strand means new DNA synthesis oneRNA primer and short growing DNA

Lagging strand also called Okazaki fragment


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DNA Replication in Eukaryotes

multiple origins of replication in eukaryotes

each origin produces two replication forks

moving in opposite direction


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Rate of Replication

In prokaryotes replicationproceeds at about

1000 nucleotides per second, and thus is done in

no more than 40 minutes.

In Eukaryotes replication takes proceeds at 50

nucleotides per second, and is completed in 60

minutes.


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DNA expression

DNA expression

involve 2 steps

Transcription Translation


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DNA to RNA to Proteins

Eukaryotes vs. Prokaryotes

In prokaryotes, transcription and translation are

coupled

translation begins while the mRNA is still being

synthesized.


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DNA to RNA to Proteins

Eukaryotes vs. Prokaryotes

In Eukaryotes,

transcription andtranslation are spatially

and temporallyseparated

transcription occurs in thenucleus to produce a pre-mRNA molecule.

pre-mRNA is processed toproduce the mature mRNA,which exits the nucleus and

is translated in the

cytoplasm.


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mRNA in Prokaryotes


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mRNA in Eukaryotes


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Transcription

occurs in four main stages:1) binding of RNA polymerase to DNA at a promoter

2) initiation of transcription on the template DNAstrand

3) elongation of the RNA chain

4) termination of transcription along with the releaseof RNA polymerase and the completed RNA productfrom the DNA template.


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Transcription1. Binding of polymerases to the initiation site at the

promoter.

Prokaryotic RNA polymerases can recognize thepromoter and bind to it directly, but eukaryoticRNA polymerases have to rely on other proteinscalled transcription factors.

2. Unwinding of the DNA double helix by helicase.

Prokaryotic RNA polymerases have the helicase

activity, but eukaryotic RNA polymerases do not. Unwinding of eukaryotic DNA is carried out by a

specific transcription factor.


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Transcription

3. Synthesis of RNA based on the sequence of theDNA template strand.

RNA polymerases use nucleoside triphosphates(NTPs) to construct a RNA strand.

4. Termination of synthesis.

Prokaryotes and eukaryotes use different signalsto terminate transcription.


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Prokaryotic promoter


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Elongation

During elongation, RNA polymerase binds toabout 30 base pairs of DNA

At any given time, about 18 base pairs of DNA

are unwound, and the most recentlysynthesized RNA is still hydrogen-bonded tothe DNA, forming a short RNA-DNA hybrid (12bp long, but it may be shorter)

The total length of growing RNA bound to theenzyme and/or DNA is about 25 nucleotides.


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Termination

Requires a termination sequence that triggersthe end of transcription.

Two classes exist:

rho dependent

a short complementary GC-rich sequence (followed by

several U residues) will form a "brake" that will help

release the RNA polymerase from the template.

rho independent.

binding of rho to the mRNA releases it from the

template.


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Termination

rho-dependent

requires a protein

called rho to bind to

specific sequence.


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Termination

rho-independent -

depends on a seqence

in mRNA which forms a

stem loop.


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Transcription in Eukaryotes

Although transcription in eukaryotes is similar to that

in prokaryotes, the process is more complex.

Instead of one RNA polymerase, there are three : RNA polymerase I(localized to the nucleolus) transcribes

the rRNA precursor molecules.

RNA polymerase IIproduces most mRNAs and snRNAs.

RNA polymerase IIIis responsible for the production ofpre-tRNAs, 5SrRNA and other small RNAs.

The mitochondria and chloroplasts have their own RNA

polymerases.


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Eukaryotic nuclear genes have three classes of

promoters which are individual for the three

types of RNA polymerases


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Termination signals end the transcription of

RNA by RNA polymerase I and RNA

polymerase III without the activity of hairpin

structures as seen in prokaryotes.

mRNA is cleaved 10 to 35 base-pairs

downstream of a AAUAAA sequence (which

acts as a poly-A tail addition signal).


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RNA processing in Eukaryotes

Messenger RNA in eukaryotes

is first made as heterogeneous nuclear mRNA /

pre-mRNA then processed into mature mRNA

through: the addition of a 5 prime cap - a guanosine nucleotide

methylated at the 7th position

addition of poly-A tails

the splicing out of introns.


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How introns are removed

The intron loops out as

snRNPs (small nuclear

ribonucleoprotein particles,

complexes of snRNAs and

proteins) bind to form the

spliceosome.

The intron is excised, and

the exons are then spliced

together.

The resulting mature mRNA

may then exit the nucleus

and be translated in the

cytoplasm.


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Translation ensures that:

polypeptide bonds are formed between adjacent aminoacid

that the amino acids are linked in the correct sequencespecified by the codons in mRNA.

mRNAs are read in the 53 direction.

Proteins are made in the amino to carboxyl direction,hence the amino terminal amino acid is added first.


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The genetic code is a set of threebase code words,called codons, in mRNA that instruct the ribosome to

incorporate specific amino acids into polypeptides.

Each base is part of only one codon.

There are 64 codons in all.

Three are stop signals and the rest code for aminoacids.


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Diagrammatic representation of tRNA

molecule


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Properties of tRNA molecule

Each kind of tRNA binds to a specific aminoacid.

It must have an anticodon, a specificcomplementary binding sequence for the

correct mRNA codon.

It must be recognized by a specific aminoacyl-tRNA synthase that adds the correct amino

acid. It must be recognized by ribosomes.


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Ribosomes Made up of 65% ribosomal RNA and 35% ribosomal proteins

arranged into small and large subunits. Ribosomes consist of two subunits (small 30S and large 50S

subunit) that fit together and work as one unit (70S) to translatethe mRNA into a polypeptide chain during protein synthesis.

The active part of the ribosome is RNA

Eukaryotes Ribosomes:

Eukaryotes have 80S ribosomes, each consisting of a small(40S) and large (60S) subunit.

Their large subunit is composed of a 5S RNA (120

nucleotides), a 28S RNA (4700 nucleotides), a 5.8S subunit(160 nucleotides) and ~49 proteins.

The small subunit has a 1900 nucleotide (18S) RNA and ~33proteins


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How rRNA transcript from DNA


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Translation


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Initiation A 30S initiation complex is formed from a free 30S subunit

plus a mRNA and fMet-tRNAf

met.

The 16S rRNA of the 30S initiation complex first base pairswith a sequence called the Shine-Delgarno sequenceupstream from the initiation codon.

This binding is mediated by IF3 with the help of IF2 andIF1.

The initiation complex then slides along the mRNA until itreaches the initiation codon.

A 30S initiation complex is formed from a free 30S subunitplus a mRNA and fMet-tRNAf

met, GTP, IF1, IF2 and IF3.

GTP is hydrolysed after the 50S subunit joins the 30Scomplex to form the 70S initiation complex.


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Shine Delgarno16SrRNA complex


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Elongation

Elongation of translation occurs in every 3codon.

When ribosome move along mRNA , amino

acid was cleaved from first tRNA andtransfered into amino acid that attach second

tRNA

The tRNA without amino acid then released.

The second amino acid then transferred into

third and so on until reach termination codon


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Elongation


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Termination

The synthesis of the polypeptide chain is terminated by releasefactors that recognize the termination or stop codon at the end ofthe coding sequence.

Prokaryotic translation termination is mediated by three factors:RF1, RF2 and RF3. RF1 recognizes UAA and UGA. RF3 is a GTP-binding protein that facilitates binding of RF1 and RF2 to the ribosome.

The release factors release the newly formed protein, the mRNA,and the last tRNA used,

The ribosome dissociates into its two subunits, which are thenreused.

Initiation in Eukaryotes


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Initiation in Eukaryotes The initiation factors are also different than those in prokaryotic

initiation:

eIF3 binds to the 40S ribosomal subunit and inhibits itsreassociation with 60S subunit

eIF6 binds to the 60S subunit and blocks its reassociation with 40Ssubunit.

eIF2 is involved in binding Met-tRNAimet to the ribosome.

eIF5 encourages association between the 43S complex(comprising the 40S subunit plus Met-tRNAimet) and large

ribosomal subunit


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Termination in Eukaryotes

Eukaryotes have 2 release factors:

eRF1 that recognizes all three termination codons

eRF3, a ribosome-dependent GTPase that helps

eRF1 release the finished polypeptide.

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