Genetics, Lecture 13 (LEcture Notes)

8/8/2019 Genetics, Lecture 13 (LEcture Notes)

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120Translation of mRNANabeelAya Al-Nobani10/11/201


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Genetics Lecture 13Wednesday, 10/11/2010Done By: Aya Al-Nobani

Translation of mRNA

Today, we'll start talking about protein synthesis (translation ofmRNA) and Doctor Zyad will continue with this subject after Eid.

* The outline of the upcoming lectures:1. The different requirements of protein synthesis.

2. Some characteristics of mRNA.

3. Structure of ribosomes and polysomes.4. Polarity of protein synthesis.

5. Function of transfer RNA (acts as an adaptor).

6. Activation of amino acids which are the building blocks of

polypeptides.

7. Amino acyl tRNA synthetase (the enzyme that activatesamino acids and charge tRNA).

8. The genetic code.

9. Codon-anticodon interactions & structure.

10. Initiation and stop codons in prokaryotes vs. eukaryotes.

11. Reading frame.

12. Mutations affecting translation thus affecting the readingframe.

Please refer to the objectives of the lectures in the slides andmake sure you understand each and every one of them.

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* Now the lecture begins

(Slide 4)This is the structure of a eukaryotic mRNA that is ready for

translation, so its capped by a 7-methyl guanine and twoother methyl groups on the first and the second nucleotides.

Then, theres the 5 untranslated region which is important,because sometimes it has sequences that control thetranscription and translation. After that, there's an importantsignal for the initiation of translation in eukaryotes andprokaryotes; the initiation codon (AUG); without this signal,translation will not be initiated.

The translation process will continue until it faces anothersignal which is the terminationcodon (UGA); (note: there are3 different termination codons or subcodons); the function ofthis codon is to stop the translation process of mRNA. Then ithas the 3 untranslated region, and then the poly(A) signaland the poly(A) tail.

So the region that will be translated is the one between theinitiation and the termination codons, meaning that amino

acids (the building blocks of protein) will be formed into apolypeptide chain according to this sequence of nucleotides.

Remember that this is the third step of the dogma of molecularbiology (the first two were replication & transcription).

* The Tools of Translation:1. mRNA2. Ribosomes

3. tRNA4. Amino acids as building blocks

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5. EnergySo we are going to talk about these components in details:

Ribosomes (slide #5):

1.Prokaryotic ribosome- Small subunit: 30S subunit = 16S rRNA + 21 proteins- Large subunit: 50S subunit = 23S rRNA + 5S rRNA + 35proteins

* The assembly of the two subunits forms the whole ribosome(the 70S ribosome).

2. Eukaryotic ribosome

- Small subunit: 40S subunit = 18S rRNA + 33 proteins- Large subunit: 60S subunit = 28S rRNA + 5S rRNA + 5.8SrRNA + 49 proteins

* The assembly of the two subunits forms the whole ribosome(the 80S ribosome).

So ribosomes are nucleoproteins; they are composed of RNAand proteins. The number of the subunit followed by (S)

represents the density or the segmentation rate of eachsubunit. Note that the assembly of the whole ribosome from thetwo subunits is not an additive process, because it representsdensity, segmentation, conformational change, and biochemicalprocesses, so its not simple math.

* The 16S rRNA in prokaryotes has a special importance, why?!>> It binds to the Shine-Dalgarno sequence, if you knowabout it!

* Steps of the Translation Process1. Initiation: starts by the assembly of the small and the largesubunits to form the whole ribosome which will search for theinitiation codon (AUG) on the mRNA.

2. Elongation: in this stage, the genetic information in themRNA will be expressed as amino acids hooked to each otherby peptide bonds, in the 5- 3 direction.

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3. Termination: once the ribosome reaches the terminationsignal (UGA), there will be dissociation of the two subunits, andthe newly synthesized polypeptide chain will be released.* Note: the direction of translation is the same as the direction

of transcription and replication (5-3), and the direction oftranslation according to the newly synthesized protein is fromthe N terminal to the C terminal.

As you see in slide #6, many ribosomes can work on the samemRNA at the same time, so you can see many stages oftranslation on the same mRNA by different ribosomes, and thisis called polysomes: many ribosomes translating the samemessage.

Q: Do all ribosomes produce the same protein on thesame mRNA?

Yes. Its the same mRNA, so all the ribosomes working on itmust produce the same protein unless mutations occur to themessage. Keep in mind that alternative splicing producesdifferent mRNAs but each mRNA will be translated into oneprotein.

tRNA(slide #7)Its very important to the translation process; it acts as an

adaptor molecule since each tRNA will bind to a specific aminoacid and to the codon on the mRNA, and so they will be themean that arrange amino acid according to the genetic code onthe mRNA, in order to form peptide bonds between them.

* Regions of tRNA1. 3 stem acceptor (which ends with CCA in all tRNA): via

this region, the amino acid binds to the adenosine (A)molecule. Every amino acid has at least one tRNA, andthere is a specific enzyme to help the amino acid to bindwith its specific tRNA.

2. The anti-codon region which interacts with the codon on

the mRNA.

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So, according to the codons on the mRNA, the needed aminoacid will bind to its specific tRNA via the adenosine nucleotideat the 3 end and this will be facilitated by the enzyme aminoacyl tRNA synthetase. This enzyme will lead to the activationof the amino acid in order to be coupled with the tRNA. So theamino acids must be first activated in order to be used asbuilding blocks for proteins.

Without the unique secondary and tertiary structures of the

tRNA, it will not be able to recognise the codon on the mRNAnor its specific amino acid, nor will it be recognised by the tRNAsynthetase, so the secondary and tertiary structures of thetRNA are very important for the proper function of the tRNA.

Q: How is tRNA synthesized?tRNA is synthesized by transcription of specific genes (there arespecific genes for each type of RNA) using RNA polymerase. Forexample: rRNA will be synthesized from one gene as one big

molecule, and then this big molecule is cleaved into pieces togive different types of rRNA: like 5S, 16S, 23S or 28S.

As you know, the building blocks of proteins are amino acids. Inorder for the amino acid to be used, it has to be activated; ithas to become acyl amino acid. This activation process servesto make the amino acid suitable as a substrate for the enzyme(tRNA synthetase). Also, it provides it with the needed energyfor binding with tRNA, so amino acyl tRNA synthetases areenzymes that charge amino acids to their specific tRNAs(couple them). Every amino acid has its specific amino acyl

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The acceptor

Anti-codon


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tRNA synthetase, so there will be at least 20 types of thisenzyme (and at least 20 types of tRNA) to activate the 20 typesof amino acids, but in reality there are more than 70 types oftRNA molecules.

Q: Why does each amino acid require a different type ofthe enzyme?

This is because of the different structures of amino acids, andthe specificity of the enzymes (binding only to a certainsubstrate). Also, having one enzyme to activate more than oneamino acid may activate unneeded amino acids which will be awaste of energy (not economical to the cell).

There can be several isoacceptor tRNAs for the same aminoacid, and that explains why we have more than 70 types oftRNA with different sequences and even different anti-codons;(in other words, there can be more than one (some have 3, 4,5, 6) tRNA that can bind with the same amino acid and havedifferent anticodons), but all of them use the same amino acyltRNA synthetase.So although we have more than one tRNA for the sameamino acid, they all use the same amino acyl tRNA

synthetase enzyme.

The difference between the different tRNAs that bind with thesame amino acid is mainly in the anti-codon region in onenucleotide.

Each amino acyl tRNA synthetase binds to:1. amino acid2. ATP: which is required to activate the amino acid, and the

amino acid will keep that energy to help in the upcomingsteps of protein synthesis.

3. isoacceptor tRNAs

So these enzymes are molecules that could bind to differentsubstrates: amino acid, ATP and tRNA. And each substrate hasits own binding site on the enzyme so that it can catalyse theactivation and the coupling of the amino acid to the tRNA.

Q: Why is it called isoacceptor?

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Acceptor because it accepts the amino acids, and iso becausethere can be more than one tRNA for each amino acid.

This is the activated amino acid; it has an adenine that came

from the ATP and its called adenylated amino acid.

And then after activation, the same enzyme will charge andcouple this activated amino acid with the tRNA at the 3 endacceptor side, releasing AMP. The resulting substrate will be

used as a building block to synthesize the polypeptide chain(protein).

* The genetic code (slide 10)The genetic code consists of 64 triplet codons (this 64 resultsfrom 43) as 4 is the number of the types of nucleotide (A, T, C,G) and 3 is the number of nucleotides in each codon. Note thatif the codon has a less number of nucleotides, the resultingpossible number of codons will not exceed 20 which is the

number of amino acids (we should have more than 20 codon,one for each amino acid in addition to the initiation and thetermination codons).All codons are used in protein synthesis. How is that possible?Each amino acid is represented by more than one codon, andthis is called degeneracy: having more than one codon for thesame amino acid. Also, we have 3 termination codons(subcodons: UAA, UAG, UGA); you have to memorise thesecodons because if I give you a sequence of mRNA and ask you

what will be the polypeptide chain that could be synthesizedfrom the sequence, you must be able to recognise thetermination codon to know when to stop.

AUG is a very important codon; it codes for methionine. Also, itis the initiation codon for any polypeptide chain. So, allpolypeptides start with Met (AUG), although it is used internallyto code for normal methionine in a polypeptide chain. So thestarting point will be from the first AUG in the mRNA and therest will code for normal methionine. In order for this to beaccomplished, the starting AUG has a specific tRNA (called

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initiator tRNA) and a specific tRNA synthetase. All the otherinternal AUG are recognised by different tRNA (to code fornormal methionine) and different tRNA synthetase.

In prokaryotes, this methionine (the starting one) will bemodified by adding a formyl group to it, and its tRNA is calledf-met tRNA and the (f) stands for formyl.

When a prokaryote that is living in a host cell produces aprotein containing this formyl methionine, the host cell willrecognise it as a foreign body and attack it. And if the proteindoes not contain the f-met, it will not be recognised by the hostcell and antibodies will not be formed. Because of that, all

proteins produced by prokaryotes start with this formylatedmet.

Back to slide (10) 5 amino acids are specified by the first two nucleotides

only (explained later). 3 additional amino acids (Arg, Leu, and Ser) are specified

by six different codons, as we said there are some aminoacids that have one codon, other have 2, 3, 4, 5 and even

6 codons. Please refer to slide # 11.

Q: How will the tRNA recognise the amino acid?By the codon and anti-codon interaction and with the help oftRNA synthetase, as we said before, the enzyme will bind to theamino acid and the tRNA, activate the amino acid and then bindit to the tRNA which will interact via the anti-codon with thecodon of the mRNA.

* Rules concerning Codon-anticodon interactions:1. Codon-anticodon base-pairing is antiparallel: whichmeans that the mRNA and the tRNA run in opposite directions(one in the 5-3direction and the other in the 3-5direction).

* Remember that we always read the codon startingfrom the 5end. *

2. The third position in the codon is frequentlydegenerate (from degeneracy)

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3. One tRNA can interact with more than one codon, andthis goes by wobble rules:If the third nucleotide of the anticodon was C it can bind with Gor I (inosine)

A with U or IG with C or UU with A, G, or II with C, U, or A

Because of these rules, one tRNA can bind with more than onecodon. For example: one tRNA leucine can read two of theleucine codons, but it will not recognise a codon of a differentamino acid, and this explains what we said before: codons of

the amino acid are recognised by the first 2 nucleotides. So thefirst 2 nucleotides represent the original amino acid.

Note that the wobble rules deviate from the Watson-crick rulesof base pairing.

* Initiation in prokaryotes and eukaryotes* Please refer to slide #14 in order to fully understand thefollowing paragraph:

In both the prokaryotes and eukaryotes, the first AUG will serveas the starting point and will be recognised by searching by thehelp of small subunit ribosomes (which is composed of proteinand RNA molecules with shine-dalgarno sequence) will basepair with that AUG.

In the prokaryotes, any AUG codon with shine-dalgarno site canact as an initiating point regardless of its position (internally ornot). So the mRNA is called polycistronic meaning that it canproduce more than one protein.

On the other hand, in the eukaryotes, only the first AUGdownstream of the 5cap can be the initiating codon. Otherinternal AUG cannot serve as an initiating point and code fornormal methionine. So the mRNA is called monocistronicmeaning that it can only produce a single polypeptide chain.

* Reading frames:The reading frame starts with the initiating codon and goes byevery subsequent triplet to be read as a codon until reaching

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the termination codon, this is the definition of the readingframe: the whole translated region from the starting till thefinishing codon, reading all codons.

The resulting polypeptide sequence depends on the codonswithin the translated region, so itll start with met coded bythe initiating codon AUG.

Sometimes, mutations occur to the mRNA resulting in thechange of the reading frame. If a deletion or insertion of onenucleotide occurs, it will result in changing the readingsequence of triplet, so the amino acid sequence will alsochange. Check the example in slide #15 (its a disease that will

be explained later).

And the effect of the change dependson the type of mutations: Missense mutations (e.g., AGC Ser to AGA Arg):

change one of the amino acids with another, the effectof this depends on whether the new one is of the sametype as the old one, and the location of the changedamino acid (important within the active site or not).

Nonsense mutations (e.g., UGG Trp to UGA Stop): sothe translation will stop before the right place due toformation of a new stop codon, and the resultingpolypeptide chain is called truncated (shortened)polypeptide chain, shorter than the normal, for exampleif the normal protein consist of 100 amino acid, thetruncated may have 20. And the rest of nucleotideswhich were supposed to be translated will not be used.

Read through, reverse terminator, or sensemutations (e.g., UAA Stop to CAA Gln): change of astop codon into a normal codon and then the translationwill not stop where its supposed to, but continuefurther on.

Silent mutations (e.g., CUA Leu to CUG Leu) do notaffect translation, the changed nucleotide resulted inanother codon that codes for the same amino acid.

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* If the mutation was in 3 nucleotides (the whole codon wasinserted or deleted) or multiple triplets, one or more aminoacids will be added or deleted and the rest will not be affected.And if the removed amino acid had an important function in the

active site, the protein would not be functional. Otherwise theresulting protein would be functional or partially functional.

An example of a genetic disease resulting from such mutationsis the haemoglobin Wayne (3 terminal frame-shift mutation)which results from deleting of a single nucleotide (U) from thenormal sequence, and this will change the whole sequence ofthe following amino acids and the resulting haemoglobin is notproperly functional. Refer to slide #16 for details on the causing

mutation.

* The END *

Sorry for any mistakes

Thanx to all my friends :DSpecial thanx to Randa Zayed & Samah Abu

Omar

Aya Al Nobani

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Genetics, Lecture 13 (LEcture Notes)