chapter 6 genetics

7/29/2019 Chapter 6 Genetics

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Chapter 6

Proteins:

- high-molecular weight nitrogen-containing compound of complex shape and composition

- composed of macromolecular subunits called polypeptides

- polypeptides are composed of amino acids linked by peptide bonds

- peptide bonds are covalent bonds between the carboxyl group of one a.a and the amino group

of the other

Molecular Structure of proteins

1. primary structure = amino acid sequence determined by base-pair sequence

2. Secondary structure = regular folding and twisting of a portion of a polypeptide/ it's the result

of weak bonds such as electrostatic bonds or hydrogen bonds/ can be either helix (hydrogen

bonds between NH group of one a.a and the CO group of another 4 a.a far) or -pleated sheet

3. Tertiary structure = 3D structre of a single polypeptide chain (conformation)/ results from

interactions between R groups of amino acids/ Hydrogen bonds, Van der Waals forces, ionic

bonds and sulfur bridges

4. Quaternary structure = only in multimeric proteins like Hemoglobin (two 142 a.a subunits

and two 146 a.a subunits)

Note:

1. Proteins fold cotranslationally. Some proteins fold by the aid of chaperones.

2. Amino acids can be acid, basic, polar neutral and non-polar neutral.


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The Nature of the Genetic Code:

The genetic code is a triplet code

Crick and his colleagues did studies on T4 bacteriophage which infects E. Coli. They studied the

effect of mutations in mutants and revertants (mutants which undergo reversion into wild type).They used Proflavin, a chemical which either delets or adds a nucleotide.

Single mutants could revert by either:

- addition (+) or deletion (-) in the site originally mutated by deletion or addition. Addition where

deletion took place initially and deletion where addition took place initially.

- addition or deletion in nearby the first mutation site (+ for -, and - for +).

Explanation: The mutations are frameshift mutations which affect the reading frame. An

opposing mutation would restore the reading frame despite the probable presence of

unsignificant substitution mutations as a result.

Crick also tried inducing three mutations of the same type, either all + or all -

The result was wild type strains with one additional or lost amino acid in the proteins produced.

No multiple combinations worked except multiples of three. (no change in reading frame)

Conclusion: The gentetic is code is a triplet code = codons in mRNA are of three nucleotides.

Deciphering the Genetic Code

Nirenberg, Khorana and Holley used Cell free protein synthesizing systems to synthesize proteins

in vitro using mRNAs of known sequences. Four approaches were used:

1. synthetic mRNAs of same base (polyG didn't work because if folded cannot be translated)

2. synthetic copolymers of either unknown sequence or known sequence. The unknown

sequence only helped know the base composition of the codon and not the sequence. The

known sequence however helped know the sequence.

3. Ribozome-binding assay (by Nirenberg): the specific nucleotide sequence of the codon is

determined. 50 codon sequences were determined using this approach.


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Experimental details of a cell-free protein synthesizing system

- E colicells are lysed, DNase is added

- Centrifuge and collect polysomes (assemblies of ribosomes with mRNA), ribosomes, mRNA,

tRNA, enzymes (supernatant)

- Add nucleotides (ATP- GTP) and radioactive amino acids (for energy)

- Add synthetic oligonucleotides

- Incubate at 370C

- Precipitate proteins with acid, leaving amino acids in solution. Radioactivity in precipitate

indicates the amount of amino acids incorporated into newly synthesized proteins

Characteristics of the Genetic Code

1. Triplet code

2. Continuous

3. Nonoverlapping (mRNA is read successively)4. Almost universal (some changes in mitochondria, chloroplast and few organisms)

5. Degenerate (Degeneracy = Redundancy of the code)

However codon usage is not random and there is codon usage bias

6. has start and stop signals

start is signaled by AUG which codes for methionine

stop is signaled by UAG (amber), UAA (ochre) and UGA (opal)

7. Wobble occurs in anticodon:

the first base in an anticodon (5 end) can potentially pair with more than one base at the

third position (3 end) of the codon three different codons can be read by one tRNA and

thus code for the same amino acidNote: Inosine (I) is a derivative of Adenosine (A)

8. There are 61 sense codons and 3 nonsense (stop or chain terminating) codons


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Transfer RNA (tRNA):

- tRNA molecules make up 10 to 15 percent of the total cellular RNA in both prokaryotes and

eukaryotes

- The size of tRNAs is 4S and they consist of a single nucleotide strand 75 to 90 nt long (withmodified nucleotides) with a significant amount of three-D structure

- Transcribed by Pol III in eukaryotes

- Molecules of tRNA bring amino acids to the ribosomes, where the aa are polymerized into a

polypeptide chain

- In both Eukaryotes and Prokaryotes, tRNAs are synthesized as pre-tRNA molecules containing

5 - leader and 3 - trailer sequences which are later (posttranscriptionally) removed by RNAse P

(leader) and RNAse Q (trailer). Then, 5-CCA-3 is added and modification of bases occur

afterwards.

- Some eukaryotic tRNAs contain introns, which are removed by different mechanisms than

those of pre-mRNA

Recognition of the tRNA anti codon by the mRNA codon

It was proved by Ehrenstein that the specificity of codon

recognition lies in the tRNA molecule, not in the amino acid it

carries.

Structure:

- The 3 end has the a.a acceptor site (The ribose of Adenosine

binds through 2' OH or 3'OH to the Carboxyl group of a.a)

- The anticodon is in Loop II


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Aminoacylation of tRNA

There are 20 different Aminoacyl-tRNA synthetases for the different amino acids.

Mechanism:

The enzyme Aminoacyl-tRNA synthetase binds ATP and the amino acid. The enzyme hydrolyzes

ATP into AMP (requires energy)and links that AMP to the amino acid froming aminoacyl-AMP.

Next the tRNA binds to the enzyme, and the enzyme transforms the aminoacyl-AMP into

aminoacyl-tRNA. Finally the AMP and the aminoacyl-tRNA are released.


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Ribosomes:

Ribosomes consist of 2 unequally sized subunits that are complexes of rRNA and a large number

of ribosomal proteins (prokaryotes and eukaryotes).

The prokaryotic ribosome

- has a size of 70S: 2 subunits are 50S and 30S

The large 50S subunit contains 34 proteins, 23S rRNA and 5S rRNA

The small 30S subunit contains 20 proteins and 16S rRNA

Ribosomal DNA is transcribed into a single precursor rRNA by the RNA Pol that also transcribes

mRNA. The transcript is processed by RNase III to produce three separate rRNA precursors,

which quickly associate with ribosomal proteins

The eukaryotic ribosome

- has a size of 80S: 2 subunits are 60S and 40S

The large subunit contains 50 proteins and 28S, 5.8S and 5S rRNA

The small subunit contains 35 proteins and 18S rRNA

RNA polymerase I transcribes the 18S, 5.8S, and 28S rRNA sequences into a single precursor

molecule. Spacer regions are removed as the pre-rRNA molecule is processed in the nucleolus

to produce mature rRNAs .

RNA polymerase I transcribes the 18S, 5.8S, and 28S rRNA sequences into a single precursor

molecule. Spacer regions are removed as the pre-rRNA molecule is processed in the nucleolus

to produce mature rRNAs.

5S rRNA is transcribed separately (by Pol III) and imported into the nucleolus where it is

assembled with the mature rRNAs and ribosomal proteins.


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During Translation, the mRNA passes through the small subunit.

Specific sites of the ribosome binds to tRNAs at different stages of the polypeptide synthesis:

- the A (aminoacyl) site is where an incoming aminoacyl-tRNA binds

- the P (peptidyl) site is where the tRNA carrying the growing polypeptide chain is located

- the E (exit) site is where a tRNA binds on its path for the R site to leaving the ribosome

The A and P sites consist of regions of both the small and large subunits. The E site exclusively

consists of regions of the large subunit.


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Translation

Initiation:

Initiation in prokaryotes starts with:

-Binding of the 30s ribosomal unit

> AUG needed plus 8-12 nucleotides upstream = Shine-Dalgarno sequence (RBS =

Ribosome Binding Sequence on mRNA) (5'- AGGAG - 3'purine rich) (the Shine-

Dalgarno sequence binds to a sequence on the 16S rRNA of the small subunit and an

experiment which involves mutating both sequence was done to prove its

importance in translation initiation of mRNA)

-Binding of the initiator tRNA at the level of AUG

> Initiation Factor IF2 brings the fMET-tRNA.fMET to the 30s/AUG upon binding GTP.

The Methionyl-tRNA synthetase charges the tRNA with methionine and then

enzyme transformylase modifies the methionine The same synthetase is used for

two tRNAs tRNA.fMET and tRNA.Met). The rest of methionines aren't modified. In many

cases the Methionine at the beginning of the polypeptide is removed.

IF1 Blocks the A site of the ribosome tRNA goes to the P site

The 30S initiation complex is now complete (mRNA, initiator tRNA, IFs, 30S subunit).

-Binding of the 50s ribosomal unit

> Leads to GTP hydrolysis and hence the release of IFs. Full 70s Ribosomal complex is

ready.

Initiation in Eukaryotes

Similar to Prokaryotes, more complex. Major differences:

- Initiator Met not modified

- Shine Dalgarno sequence is not found. The 40S subunit (small eukaryotic ribosomal subunit)

has another way for finding the intitiation code (AUG) in Kozak sequence.

First, eIFs including CBP (Cap Binding Protein) bind to the cap at the 5' end of the mRNA. Then acomplex (40S subunit + initiator Met-tRNA + several IFs + GTP) moves along the mRNA and scans

for the AUG in Kozak sequence. AUG not in Kozak is not an initiator code. Now the 60S subunit

displaces the IFs (except eIF-4F) producing the 80S initiation complex in which the Met-tRNA is

bound to AUG of mRNA at P site. The Poly(A) tail is attached to the IFs at the cap (by an

enzyme)mRNA loops stimulates initiation.


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Elongation:

Uses Elongation Factors (Ef). We'll talk about bacteria, and it's similar in eukaryotes.

1. Binding of aminoacyl-tRNA at the A site

The aminoacyl-tRNA is brought to the ribosome bound to Ef-Tu--GTP. When the aminoacyl-

tRNA binds to the codon in the A site (by hydrogen bonds), GTP hydrolyzes into GDP releasing

the Ef-Tu--GDP. (Ef-Tu--GDP is recyclable) (Other EFs in eukaryotes are used)

2. Peptide formation bond

Two steps:

1. Cleavage of the bond between the amino acid and the tRNA at the P site. A free tRNA

is generated at site P.

2. Peptide bond is formed between the freed amino acid and the one on tRNA site A.

Later it would be between a peptidyl (site P) and an amino acid (site A). The reactionis catalyzed by ribozyme Peptidyl Transferase located between sites A and P.

3. Translocation

Translocation requires Ef-G and GTP. An EF-G--GTP complex binds to the Ribozome, and the

hydrolysis of GTP leads to translocation along the mRNA. The uncharged tRNA now binds

transiently to site E (in large subunit) and doesn't leave before the peptidyl-tRNA is bound

correctly at site P (the uncharged tRNA also prevents a new aminoacyl-tRNA from binding to site

A). Now the uncharged tRNA leaves and the cycle goes on until the ribosome translocates to a

stop codon.

In both eukaryotes and prokaryotes, once the ribosome moves away from the initiation site,

new ribosomes begin translation of the same mRNA simultaneously. This forms what is called

the polyribosome, or polysome (8 to 10 ribosomes may be present).


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Termination:

The ribosome recognizes a stop codon with the help of proteins called release factors (RF),

which have regions that read the codons.

1. In E. Coli, RF1 recognizes UAA and UAG, and RF2 recognizes UAA and UGA. The binding ofeither to a stop codon triggers Peptidyl Transferase to cleave the polypeptide chain releasing it

and leaving an uncharged tRNA.

2. Now RF3-GDP binds to the ribosome releasing RF(1 or 2) from the stop codon and ribosome.

Afterwards, GTP replaces GDP RF3 hydrolyzes GTP and releases itself from the ribosome.

3. the Ribosome recycling factor (RRF) binds to the ribosome at A site. Then the EF-G--GTP

binds and, upon hydrolysis of GTP to GDP, translocates the RRF to the P site and the uncharged

tRNA to the E site. The RFF releases the uncharged tRNA, and the EF-G releases the RFF causing

the dissociation of the ribosomal subunits.

In Eukaryotes

It's the same, but the stop codons are all recognized by one release factor called eRF1, and eRF3

stimulates the termination events (instead of RF3).

Also there is no RRF in eukaryotes although the ribosome is recycled.


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Protein Sorting in the Cell

In eukaryotes, the proteins produced are either sent to cell compartments or secreted.

In prokaryotes the proteins may stay inside the cell, localize in mmbrane or be secreted.

The sorting of proteins is directed by signal/leader sequences on the proteins being

synthesized (during translation).

Signal Hypothesis: Proteins sorted by the Golgi bind to the ER by a 15 to 30 hydrophobic

N-terminal amino acids (signal sequence).

The signal sequence produced by translation is exposed on the Ribosome. Signal

recognition particle (SRP, RNA-protein complex) binds the sequence and blocks further

translation before the growing polypeptide-SRP-ribosome-mRNA complex binds to the

ER.

The SRP binds to SRP receptor in the ER membrane which causes:

- Firm binding of ribosome to ER membrane

- Release of SRP

-Resumption of transltion

The SRP is then degraded by signal peptidase. The produced polypeptide in the ER

cisternal space is typically modified by addition of carbohydrates glycoproteins.

chapter 6 genetics

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