[WEEK6] Part 1 Gene Expression

Learning objectives

https://www.youtube.com/watch?v=8wAwLwJAGHs 

 

 

Understand and explain the central dogma of molecular biology. 

Central dogma explains the flow of the gene, which is from DNA to mRNA, and protein. 

Also, it explains that the sequence of base determines the genotype

 

Explain how RNA differs from DNA.  

RNA has deoxyribose as a sugar, DNA has ribose sugar instead

RNA is single stranded, and DNA is double stranded with hydrogen bonding between bases

RNA has Uracil instead of thymine for DNA

 

Distinguish between transcription and translation and compare where transcription and translation occur in prokaryotes and in eukaryotes.

Transcription occurs in nucleus and Translation occurs in cytoplasm for eukaryotes.

Prokaryotes has no nucleus so both of the process happen immediately

 

Define what a codon is and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide.  

Codon is made up of three pair of base sequences. It has a role as a genetic code and determine the type of amino acid. Codon is the sequence on mRNA that determines aminoacids type by grouped in three, as a genetic code

 

Explain why polypeptides begin with methionine when they are synthesised.  

Because the start codon (AUg) which stimulate the translation which  indicates the methionine aminoacid

 

Explain what it means to say that the genetic code is redundant and unambiguous.  

Redundancy of genetic code means more than one codons point out same amino acid, for example, first two base don't affect to the aminoacid type changes. Unambiguous means that one genetic code dictate one aminoacid, no more than one. 

 

Explain the significance of the wobble.  

Eventhough tRNA detects the third base differently, tRNA still brings the same amino acid. This unstandard base pairing is called Wobble. 

 

Explain how RNA polymerase recognises where transcription should begin.  

RNA polymerase for prokaryotes has sigma factor as part of them. When sigma factor binds to the pormoter region, transcription starts. All bacterial promoters have a -10 box and a -35 box, the remainder of the promoter sequence varies

 

Describe the promoter and its role, including the TATA box. 

Promoter is the primer for RNA synthesise, transcription. For Eukaryotes, promotor region is more diverse and complex. Many of the eukaryotes promoters include TATA box sequence. TATAbox is located about 30 base pairs upstream of the start point of the transcription.

 

Explain the general process of transcription. 

When a RNA polymerase unwind the RNA double helix and binds to the promoter region with the help sigma factor (for prokaryotes), and transcription factor (for eukaryotes) recognising it, it synthesise the mRNA by bringing nucleotides and binds them with phosphodiester bonding. 

 

Explain how RNA is modified after transcription in eukaryotic cells.  

It is processed by splicing, and capping and tailing. 

Splicing is the process of removing introns which is uncoding region, and binds extrons together. Unlike to prokaryotes, eukaryotes include many untriscriptional region which has a role as enhancer, silencer and so on.

small nuclear ribonucleoproteins (snRNP) form slicesome to splice the introns.

Capping is done by attaching 5' cap and poly A tail. 5' cap function as sending the signal for starting the translation

 

Describe the functional significance of introns. 

Introns have a role as helping the transcription process and affects the gene expression. Enhancers, silencers, and promoter regions.. all the regulatory gene is included as introns.

 

Describe the structure and functions of tRNA and explain how tRNA is joined to the appropriate amino acid. 

tRNA is small, 75-85 base long, and folded shaped which has anticodon attached part, and amino acid attached part.

tRNA is synthesised by the enzyme aminoacyl tRNA synthetase. When aminoacid and ATP bind to the active site of the enzyme, 2 phosphate is released and amino acid and AMP binds. Matching tRNA bonds to amino acid displacing AMP. tRNA leaves the enzyme with the amino acid charged

 

Describe the structure and functions of ribosomes.  

Ribosome is made out of rRNA and protein. It has large subunit and small subunit. when mRNA binds to the small unit of mRNA, tRNA come and binds to the startcodon and the large subunit binds to them. Large subunit has E,P, A site to receive incoming tRNA.

 

Describe the process of translation (including initiation, elongation, and termination) and explain which enzymes and energy sources are needed for each stage. 

Initiation is started by mRNA binding to the small ribosomal subunit. Charged tRNA carrying methionie attaches to the P site of the small subunit, and after large ribosomal subunit binds and form initiation complex.

Elongation starts when tRNA comes to the A site and peptide bond forms between amino aicd of those tRNA placed in P and Asite. tRNA in the P site moves to E site and new charged tRNA comes to the A site to elongate peptide.

When a release factor binds to the E site, which looks similar to tRNA but has no aminoacid attached to it, the translation terminates. Release factor hydrolysis of the bond linking the tRNA (in the P site) with the polypeptide chain

 

Define a gene and describe its components and their role in gene expression

Gene is a segment of chromosome. It is made up of base sequences that determine the characteristic of the organism. It also includes the non-transcriptional sequence that helps the gene expression. (functional sequence)

 

Compare and contrast silent, missense and nonsense mutations. 

Silent mutation occurs by changing the last sequence of base pair. Because the first two pairs of bases is all same between the genetic code that indicating the common aminoacid, it doesn't bring about any mutations. Missense mutation is caused by bringing incorrect amino acid. Nonsense mutation occurs by charging STOP codon so the translation stops.

 

PPT

 

 

 

WHAT IS GENE EXPRESSION

*Transcription : synthesis of RNA from a DNA template

*Translation : synthesis of a protein from the mRNA template

*Ribosomes are the sites of translation 

=> This flow of information within the cell is summarised in the CNETRAL DOGMA

 

 

THE CENTRAL DOGMA

-DNA replication is also included in the CENTRAL DOGMA

*GENOTYPE is determined by the sequence of bases in DNA

EXCEPTIONS to the CENTRAL DOGMA

: tRNA , rRNA (which are not function as translated into protein)

: sometimes information flows in the opposite direction (RNA to DNA)

 

Reverse transcriptase 

 

 

 

 

EXPERIMENT

Archibald Garrod

George Beadle & Edwaed Tatum

Class 2 mutants was lack of enzymeB, which is for onrnithine. As they don't have enzyme for ornithine, they couldn't grow in MM + ornithine condition

 

 

 

 

 

 

 

 

 

DNA AND RNA STRUCTURE

DNA RNA

-Double helix single strand

-Phosphate, deoxyribose backbone, Nitrogenous base facing inward  ribose

-Hydrogen bonding between bases, phosphpdiester bonding between nucleotides

-3'->5', 5' ->3' antiparallel

-A=T, G=1C (purine A,G with 2rings, pyrimidines TC with 1 ring) Uracil instead of thymine

 

 

 

 

 

 

 

 

 

TRANSCRIPTION

WHY aren't proteins translated directly from DNA?

-To protect DNA

-More copies of a protein can be made simultaneously by multiple RNA transcription, with multiple RNAs

-Each RNA transcript can be translated repeatedly

 

RNA is made up of single strand of DNA -> Template strand

The other strand -> non-Template, Coding strand  (It has matching sequence of mRNA except for T and U)

-Bacteria have only one type of RNA polymerase

-Eukaryotes have RNA polymerase 1,2,3

-RNA polymerase 2 synthesise mRNA

-Unlike DNA polymerase, RNA polymerase doesn't require Primer

-RNA polymerase transcipt in 5'->3' direction

 

 

*Hydrogen bonds form between complementary base pairs 

*Phophodiester bond is formed by RNA polymerase after base pairing occurs 

-Promotor : specific nucleotide sequence which is located near the start of the gene, where RNA polymerase can recognise

 

-When DNA replication, primerase synthesise RNA primer to indicate the start point for DNA polymerase

-For Transcription, RNA polymerase doesn;t need primer, it recognise certain area of the DNA, which is called primer

(For bacteria, -10 and -35 box / For eukaryotes, TATAA box)

 

*Prokaryotic RNA polymerase is a holoenzyme made up of the core enzyme, which has the ability to synthesise RNA, and a sigma subunit

*Sigma acts as a regulatory factor, guiding RNA polymerase to specific promotor sequences on the DNA template strand

*All bacterial promotors have 10 box and 35 box the remainder of the promotor sequence varies

 

*Holoenzyme

 

 

EUKARYOTES

*Eukaryotes have much more diverse and complex promotor series

*Many of the eukaryotic promotors include TATA box (TATAA or similar) centered about 30 base pairs upstream of the transcription start site

*BASAL TRANSCRPTION FACTORS bind to the DNA promotor, initiation transcription

(perform a similar function to bacterial sigma proteins)

(basal transcription factors include many proteins, they are not holoenzyme)

*In eukaryotes, the product of transcription should be processed before translation started

 

PROKARYOTES

*Prokaryotes have no nucleus segregating transription from translation, and can transcribe and translate the same gene simultaneously. The new protein quickly diffuses to its operating site

*Functional mRNA, noncoding regions must be removed

*Exons are the coding regions (part of final mRNA product)

*Introns are the intervening noncoding sequences (NOT a part of final mRNA product)

=> As a result actual functional mRNA is much smaller than actual Eukaryotic genes

 

THEN what is the function or purpose of introns or other parts in DNA that producing part of non functional mRNA ????

SPLICING 

*PRIMARY RNA TRANSCRIPT -> contain exons and introns

*small nuclear ribonucleoproteins (snRNPs) form a complex called spliceosome

SMALL NUCLEAR RIBONUCLEOPROTEINS

*5' cap : serves as a recognition signal for the translation machinery

*Poly (A) tail : extends the life of an mRNA by protecting it from degradation

 

 

 

 

 

 

 

 

 

 

 

 

 

TRANSLATION

*TRIPLET CODE 

: a series of non-overlapping, three nucleotide words called a codon

* Association between CODON and AMINOACIDS -> genetic code

 

GENETIC CODES are..

1. redundant but not ambigious

(multiple triplet codes indicate to single aminoacids, but one triplet code-> one aminoacid this matching is clear)

2. nearly universal, shared by the simplest bacteria to the most complex animals

 

 

*tRNA can recognise more than one codon for a particular a'a

This none standard base-pairing is termed WOBBLE

so the sequenc ( base sequence of DNA ) is different by different animals

BUT the genetic code is same, 

RIGHT?

*RIBOSOMES are named based on their sedimentation rate 

-Eukaryote : 60S and 40S

-Prokaryote : 50S and 30S

(In eukaryotes, the subunits are made in the nucleus)

 

*A site : acceptor site for an aminoacyl tRNA

*P site : peptide bond forms that adds an amino acid to the growing polypeptide chain

*E site : where aminoacid exit the ribosome

tRNA

-each carries a specific amino acid , and anticodon on the other end

-tRNA binds to the ribosome

ENZYMES

-AATS (Aminoacyl tRNA synthetase) : charges tRNA's attaches correct amino acid to its tRNA

(THE WAY TO MAKE tRNA attached with aminoacid)

1. amino acid and ATP binds to aminoacyl tRNA

2. ATP loses two P groups -> AMP (AMP and amino acid are bind to aminoacyl tRNA synthetase)

3. tRNA covalently bond to aminoacid, displacing AMP

4. tRNA with amino acid attached released by the enzyme

-> As a result, tRNA with anticodon one end, amino acid one end is formed !!!

 

-Peptidyl transferase : catalyse bonds between amino acids in growing polypeptide chain

ATP and GTP for energy

 

*energy requirements

-4P 

-2P : during formation of aminoacyl tRNA

-2P : bonds from GTP during binding of aminoacy tRNA to A site and translocation

-Extra GTP for initiation complex termination

 

*Translation Base-pairing

1) mRNA and tRNA -> codon and anticodon

2) mRNA and small ribosomal subunit

3) tRNAs and ribosomal subunits at A and P sites

 

*What is GTP?

TRANSLATION

<INITIATING TRANSLATION IN BACTERIA> 

1) mRNA binds to small subunit of ribosome

2) f-MET tRNA binds 

3) Large subunit binds

 

1. mRNA binds to the small ribosomal unit. Charged tRNA (carrying methionine) attaches to the start codon at the P site on the small subunit

2. Large ribosomal subunit complexes with mRNA/tRNA forming the initiation complex

 

<ELONGATION OF POLYPEPTIDES>

1) incoming aminoacyl tRNA

2) peptide bond formation

3) translocation

4) Incoming aminoacyl tRNA

5) Peptide bond formation

6) Translocation

 

1. An aminoacyl tRNA binds to the codon in the A site via complementary base pairing between anticodon and codon

2. Peptide bonds form between amino acids on the tRNAs in the P and A sites

After peptide bond formation, the polypeptide on the tRNA in the P site is transferred to the tRNa in the A site

3. Translocation occurs when elongation factors move the mRNA down the ribosome three nucleotides at a time, and the tRNA attached to the growing protein moves into the P site.

The A site is now available to accept a new aminoacyl tRNA for binding to the next codon. The tRNA that was in the P site moves to the E site, and if the E site is occupied, that tRNA is ejected

 

<TERMINATION>

1) Release factor binds to stop codon

2) Polypeptide is released

3) Ribosome subunits seperate

 

1. The termination phase starts when the A site encounters a stop codon

2. This causes a protein called a release factor to enter the site. Release factors resemble tRNAs in size and shape but do not carry an amino acid

3. These factors catalyse hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain

 

*Post-translational modification -> before they are ready to go to work in a cell, they should go through extensive series of processing steps

 

*Molecular chaperones speed folding of the protein, folding determines a protein's shape and therefore its function

 

*Many proteins are altered by enzymes that add or remove a phosphate group. THeses changes often switch the protein from an inactive state to an active state or vice versa

 

*SO THE sequence of base determines the aminoacid chain made in translation process, and also after the translation, folding and the shape of protein also determine its function!

 

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SUMMARY

 

1) TRANSCRIPTION 

(RNA is transcribed from a DNA template)

 

2) RNA PROCESSING 

(In eukaryotes, the RNA transcript (pre-mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm)

 

3) FORMATION OF INITIATION COMPLEX

(5’cap and polyA)

(After leaving the nucleus, mRNA attaches to the ribosome)

 

4) AMINO ACID ACTIVATION

(Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP)

(Aminoaciyl t RNAsynthetase makes amino acid tRNA)

 

5) TRANSLATION

(A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time)

(When completed, polypeptide is released from the ribosome)

 

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<INITIATING TRANSLATION IN BACTERIA> 

1) mRNA binds to small subunit of ribosome

2) f-MET tRNA binds

3) Large subunit binds

 

1. mRNA binds to the small ribosomal unit. Charged tRNA (carrying methionine) attaches to the start codon at the P site on the small subunit

2. Large ribosomal subunit complexes with mRNA/tRNA forming the initiation complex

 

 

<ELONGATION OF POLYPEPTIDES>

1) incoming aminoacyl tRNA

2) peptide bond formation

3) translocation

4) Incoming aminoacyl tRNA

5) Peptide bond formation

6) Translocation

 

1. An aminoacyl tRNA binds to the codon in the A site via complementary base pairing between anticodon and codon

2. Peptide bonds form between amino acids on the tRNAs in the P and A sites

After peptide bond formation, the polypeptide on the tRNA in the P site is transferred to the tRNa in the A site

3. Translocation occurs when elongation factors move the mRNA down the ribosome three nucleotides at a time, and the tRNA attached to the growing protein moves into the P site.

The A site is now available to accept a new aminoacyl tRNA for binding to the next codon. The tRNA that was in the P site moves to the E site, and if the E site is occupied, that tRNA is ejected

 

<Termination>

1) Release factor binds to stop codon

2) Polypeptide is released

3) Ribosome subunits seperate

 

1. The termination phase starts when the A site encounters a stop codon

2. This causes a protein called a release factor to enter the site. Release factors resemble tRNAs in size and shape but do not carry an amino acid

3. These factors catalyse hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain

 

 

 

GENE STRUCTURE AND MUTATION

*Chloramphenicol : binds to 50S r-RNA and inhibits formation of peptide bond

*Erythromycin : binds to 50S r-RNA and prevents movement along m-RNA

*Tetracycline : interfers with the t-RNA anticodon reading or m-RNA codon

*Stretomycin : changes shape of 30S r-RNA and causes m-RNA to be read incorrectly

 

 

GENE 

: segment of DNA in a chromosome which consist of not transcribed promotor region and transcribed coding segment

: transcription product is either a functional RNA (tRNA or rRNA) or an mRNA which is translated to a polypeptide

: the entire nucleic acid sequence needed for the synthesis of an RNA sequence or functional polypeptide

 

DNA sequence changes

1. Substitution of one base for another (point mutation) -> sickle cell disease

2. Insertion or deletion of bases -> frame-shift mutation 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SPQ

-DNA vs RNA

: deoxyribose / ribose

: Thymine base / Uracil base

: double stranded / Single stranded

1. Initiation

-mRNA binds to the small subunit of ribosome

-tRNA with methionine binds to the P site of the small subunit, mRNA's codon

-Large subunit ribosome forms the initiation complex

 

2. Elongation

-aminoacyl tRNA binds to the A site, amino acids form peptide bonds with the amino acid of the tRNA in the P site.

-Peptidyl transferase activity of ribosome.

-The ribosome moves by 1 codon, translocation occurs.

-tRNA which moves to E site released from ribosome and new aminoacyl tRNA binds to the codon of mRNA, A site.

 

3. Termination

-When ribosome reachs to the stop codon (UAA, UAG, UGA), release factor which ressemble to the tRNA but has no amino acid binds to the A site and and the protein chain and all components are released

 

Transcription

RNA polymerase

It is made up of nucleotide, which has deoxyribose, phosphaste and nitrogenous base.

Base includes Adenine, Thymine, Guanine and Cytosine. 

Bases form hydrogen bond and phosphate and deoxyribose form phosphodiester bond to form back bone of the DNA. Deoxyribose has phosphate group in 5' end and OH group in 3' end, and base attached to 1' end. DNA is antiparallel double strands (One is 3' ended, and the other is 5' ended)

 

5.

Genetic code is three base pairs that is encoded to synthesis protein in translation process.

They are 

-unambiguous : one genetic code pairs to one amino acid 

-redundant : one amino acid can be encoded by multiple genetic codes 

-universal : Almost all organisms share same genetic codes 

 

1 start codon (AUG), 3 stop codon (UAA, UAG, UGA), and 60 others => 20 amino acids

 

6. 

Promotor is a region of sequence of DNA located in the upstream of the coding gene region

RNA polymerase starts transcription from the promotor region

Promotor region is recognised by sigma protein in prokaryotes, and basal transcription factors in eukaryotes

 

7.

-From the promotor, to gene coding region is downstream direction

-From the promotor, opposite to gene coding region is called upstream direction

 

8.

a. RNA is single stranded

b. DNA has thymine not uracil

c. OH group is attached to 3' carbon

e. RNA can be also found in cytoplasm during translation

9.

DNA, after transcription, produces tRNA, mRNA, and rRNA

mRNA before processed, it includes exons and introns

 

10.

DNA polymerase : primer o, helicase o, exonuclease activity o , proof reading o

RNA polymerase : primer x , promotor region o, helicase x, exonuclease activity x, proof reading x

 

A) RNA polymerase also uses DNA as a template

B) RNA polymerase binds to double stranded DNA, it then seperates the double strand. It is able to seperate the double strand of DNA

DNA polymerase binds to single strand DNA, which is already seperated by helicase

C) DNA polymerase is more accurate as it has ability to proof read and do exonulcease activity

D) RNA polymerase can bind to promotor region (part of template) in the DNA with sigma protein/ basal transcription factor and starts its synthesis  BUT DNA polymerase requires primer (part of new strand) to starts synthesis

E) 

RNA polymerase need to seperate DNA double strand to synthesis RNA copy. BUt it doesn't need helicase

11. Prokaryotic RNA doesn't have introns, so it doesn't need to go onto splicing process, which is removing introns and connecting exons together

13.

Because Exon is the region directly related to matching corresponding amino acid

14.

Frame shift mutation -> one deletion or insertion of base changes the reading frame

D) It has E deleted but rest of them are same + reading frame (3 pairs) are changed

C) single substitution -> T changed into R