Translation doc tengco

8/8/2019 Translation doc tengco

1/65

TRANSLATION : PROTEIN

SYNTHESIS AND THE GENETICCODE

G.S.B.C. TENGCO, MD


2/65

LEARNING OBJECTIVES :

EXPLAIN the importance of studying protein

synthesis

DEFINE the following terms :

translation codons

genetic code

EXPLAIN the genetic code, its basic characteristics

and the principle of recognition

EXPLAIN the Wobble Hypothesis and its importance


3/65

DISCUSS the steps in amino acid activation together

with the role of tRNA and aminoacyl tRNA

synthetase

DESCRIBE the following steps in protein synthesis

including the role of enzymes and factors in each

process:

initiation, elongation and termination DIFFERENTIATE protein synthesis in prokaryotic and

eukaryotic cells


4/65

DISCUSS the role of signal peptides and the roughendoplasmic reticulum

DESCRIBE the different methods of posttranslational

modification of proteins DISCUSS the mode of action of some antibiotics

affecting bacterial protein synthesis

DISCUSS the effects of some viruses in host translation

and the role of Interferons DESCRIBE the energy expenditure for translation

DISCUSS the regulation of translation


5/65

The Central Dogma of Genetics


6/65

VIRAL REVERSE TRANSCRIPTASE or

RNA DIRECTED DNA POLYMERASE


7/65

DEFINITION OF TERMS

TRANSLATION

- is the conversion of the genetic code (nucleotide

sequence of an mRNA) into the amino acid

sequence of a protein

- is cytoplasmic in eukaryotes

CODON

-three-letter code words for amino acids

GENETIC CODE

- linear array of codons


8/65

4 nucleotides 20 amino acids

42 = 16 < 20 amino acids

43 = 64 > 20 amino acids

Thus :

61 codons code for the 20 amino acids3 are nonsense codons


9/65

FURTHER INVESTIGATIONS

SHOWED :

Tobacco necrosis satellite virus has RNA with 1200nucleotides which code for 400 coat proteins

Therefore : 1200/400 = 3 In bacteriophage T4 :

- deletion/insertion of 1 or 2 nucleotides resulted tosynthesis of wrong amino acids and defective coat

proteinsBUT :

- deletion/insertion of 3 consecutive nucleotides = onlyone amino acid residue missing or added


10/65

LATER EXPERIMENTS PROVED THAT :

UUUUUUUUU = PhePhePheCCCCCCCCC = ProProPro

AAAAAAAAA = LysLysLys

CONCLUSION :

The genetic code is in the form of

TRIPLETS/CODONS


11/65

THE GENETIC

CODE


12/65

IMPORTANCE :

KNOWLEDGE OF THE GENETIC CODE EXPLAINS :

the basis of most genetic diseases such as mutations

How viral infections disrupt host protein synthesis The mechanism of action of some antibiotics is the

inhibition of protein synthesis of bacterial cells

(selective toxicity)


13/65

CHARACTERISTICS OF THE

GENETIC CODE

DEGENERATE - an amino acid may have more

than one codon

- degeneracy resides on the third nucleotide of the

triplet

Ex: Ser 6, Val 4, but Trp and Met 1

UNAMBIGUOUS - a codon codes for only onespecific amino acid and no other

Ex: UUU - is only for Phe


14/65

NONOVERLAPPING - message is read in a

continuous manner with no punctuations until a

termination signal appears

Ex: AUGCUUGCCAAACCCAGUUAA

Met Leu Ala Lys Pro Ser Stop

UNIVERSAL - except in some mitochondrial codons


15/65

OTHER FEATURES

Does not change with age or geographical location

INITIATING CODON - is AUG which codes for Met THREE TERMINATING CODONS - UAA, UAG and

UGA (nonsense codons)

Reading of the codons is from 5 to 3

Synthesis of protein is from N to C terminal


16/65

PROBLEM :

Amino acids have no direct or special affinity with

nucleic acids

THIS IS SOLVED BY :


17/65

TRANSFER RNA (tRNA) : the adapter

molecule

adapter into which the amino acid is plugged to

be adapted into the nucleotide language of the

genetic code - F. H. Crick

One tRNA exists for each of the 20 amino acids

Each specific tRNA is charged with its specific amino

acid

3 acceptor arm binds the amino acid Anticodon arm base pairs with codon in antiparallel

fashion


18/65


19/65

But what mechanism sees to it that the proper aminoacid is bound to its proper tRNA?


20/65

AMINOACYL TRNA SYNTHETASES

Combines the proper amino acid with its proper

tRNA at the expense of ATP

Must be HIGHLY SPECIFIC

Recognizes only L-amino acids with free -amino

group

One synthetase exists for each amino acid but is

capable of recognizing all tRNA acceptor for eachamino acid


21/65


22/65

tRNA


23/65

codon

5_________________________________3

G C U G C C G C A

C G I C G I C G I

3_________________________________5

anticodon

The single anticodon IGC recognizes the 3 codons of

Alanine


24/65

DEGENERACY OF THE GENETIC

CODE

First two letters are very specific

Degeneracy rests on the third letter/nucleotide, the

Wobble position

Allows for nonstandard base pairing on the third

position

Thus, a single tRNA can recognize several codons

not necessary to have 61 tRNAs to recognize the

61 code words (31 cytoplasmic tRNAs, 22

mitochondrial)


25/65

DEGENERACY :

- Facilitates rapid codon-anticodon

unbinding/dissociation

- Is protective for survival value

- Ex:

UCA - codes for Ser

Mutation to UCG - is also a codon for SerResult is : Silent mutation


26/65

STEPS IN PROTEIN SYNTHESIS

Initiation

Elongation

Termination


27/65

REQUIREMENTS FOR PROTEIN

SYNTHESIS

+ GTP


28/65

INITIATION

Steps in Initiation :

Ribosome dissociates into 40s and 60s subunits

Binding of ternary complex ( met-tRNA, GTP and

eIF2) to 40 s ribosome 43s PREINITIATION COMPLEX

Binding of mRNA to 43s preinitiation complex to form

48s INITIATION COMPLEX

Combination of 48s initiation complex with 60s

ribosomal subunit to form 80s INITIATION COMPLEX


29/65

INITIATION


30/65


31/65


32/65


33/65

POSITIONING OF INITIATING

CODON


34/65

KOZAK CONSEQUENCE

SEQUENCE

Helps locate the correct AUG initiation codon

-3 -1 +4

GCCA/GCCAUGG


35/65

CODON-ANTICODON FOR

INITIATING AMINO ACID METHIONINE


36/65

ELONGATION

Steps in elongation

Binding of the specified aminoacyl-tRNA to the A site

Peptide bond formation between -amino group of

new aminoacyl-tRNA in A site with esterified caboxylgroup of the peptidyl-tRNA on the P site catalyzed by

PEPTIDYLTRANSFERASE

Translocation of the newly-formed peptidyl-tRNA from

A site to P site


37/65

ELONGATION


38/65


39/65


40/65

SECOND SPECIFIED AMINO ACID

ENTERS THE A SITE


41/65

PEPTIDE BOND FORMATION BY

PEPTIDYLTRANSFERASE


42/65

TRANSLOCATION


43/65

TERMINATION

mRNA termination signal ( either UAA, UAG or

UGA ) enter the A site

Releasing factor recognizes termination signal

arriving at A site

Peptide bond from tRNA in P site is hydrolyzed by

Peptidyltransferase

Release of protein and tRNA from P site Ribosome again dissociates into 40s and 60s for

another cycle


44/65


45/65

RECOGNITION OF TERMINATING

SIGNAL


46/65

SIGNAL PEPTIDE OR LEADER

SEQUENCE

Are extra amino acids at the N terminal of some

proteins

15 30 hydrophobic amino acid residues which can

interact with nonpolar portions of membranes

A signal for the selection and binding of proteins

destined for EXPORT OR SECRETION Ex: insulin, albumin, collagen, immunoglobulins and

some viral coat proteins


47/65

POLYRIBOSOMES/POLYSOMES


48/65

Export proteins for secretion are synthesized by

membrane bound ribosomes

Ex: hormones, digestive enzymes, albumin

Local proteins for intracellular use are synthesized

by free ribosomes

Ex: intracellular enzymes, hemoglobin inreticulocytes


49/65

ENERGY CONSUMPTION PER PEPTIDE

BOND FORMED

Charging of the tRNA

with aminoacyl group 2 (ATP AMP)

Entry of aminoacyl tRNA

A site 1 (GTP GDP)

Translocation of peptidyl

tRNA from A site to P site 1 (GTP

GDP)4 high energy bonds

per peptide bond formed


50/65

REGULATION OF TRANSLATION

Both occur at the level of INITIATION

1. phosphorylation of eIF-2A causes it to bind

tightly to GTP-GDP recycling protein

BLOCKS PROTEIN SYNTHESIS BY PREVENTING THE

FORMATION OF 43S PREINITIATION COMPLEX

- a host defense against viruses

- may also help conserve energy during stress andnutrient deprivation


51/65

2. Phosphorylation of 4E :

A. ACTIVATION - 4E binds more avidly to

mRNA cap enhances protein synthesis

- stimulated by INSULIN and mitogenic growth

factors

- Therefore, INSULIN PROMOTES PROTEINSYNTHESIS in liver, muscle and adipose tissue


52/65

B. SOME PROTEINS MAY INACTIVATE 4E

- BP1 (or BP2 or BP3) binds to 4E to prevent it frombinding to 4G to form 4F 4F cannot bind to 40s

subunit INHIBITION OF TRANSLATION- effect may be REVERSED BY INSULIN byPHOSPHORYLATION OF BP1

*RATE LIMITING STEP IN TRANSLATION :Recognition of mRNA cap by 4E


53/65


54/65

POSTTRANSLATIONAL MODIFICATION

AND PROCESSING

Removal of signal peptide or amino acid terminal -procollagen to collagen

Formation of disulfide bridges and removal ofconnecting ( C ) peptide insulin

Hydroxylation - hydroxyproline and hydroxylysine ofcollagen

Methylation methyllysine of muscle proteins

Glycosylation - glycoproteins

Phosphorylation - phosphoserine of glycogenphosphorylase

Acetylation


55/65

PROKARYOTIC AND EUKARYOTIC

PROTEIN SYNTHESIS

Translation starts even

before transcription isfinished

No further modification

Ribosome is 70s

Initiating amino acid isformylmethionine

mRNA is polycistronic

Compartmentalizationseparates transcription from

translation Extensive modification

Ribosome is 80s

Initiating amino acid ismethionine

mRNA is monocistronic*cistron smallest unit of genetic

expression

PROKARYOTES EUKARYOTES


56/65

ANTIBIOTICS THAT INHIBIT PROTEIN

SYNTHESIS

Antibiotics become useful only if they exhibit SELECTIVE TOXICITY

Specifically inhibits bacterial protein synthesis by acting only on 70sribosomes

CHLORAMPHENICOL, CLINDAMYCIN - inhibits ribosomalPEPTIDYLTRANSFERASE preventing peptide bond formation, binds to 50s

subunit ERYTHROMYCIN - prevents translocation

STREPTOMYCIN - distorts A site thereby initiates misreading of code

TETRACYCLINE - prevents binding of aminoacyl-tRNA to A site 30s subunit

RIFAMPICIN inhibit RNA Polymerase

QUINOLONES inhibit replication thru inhibition of DNA gyrase AMINOGLYCOSIDES irreversibly binds to 30s subunit

SULFAS inhibits PABA thereby inhibiting folic acid synthesis in bacteria


57/65

SOME ANTIBIOTICS AND BACTERIAL

PROTEIN SYNTHESIS


58/65

SIMILARITY OF SULFAS TO PABA


59/65

INHIBITORS OF ANIMAL PROTEIN

SYNTHESIS

DIPHTHERIA TOXIN - catalyzes ADP ribosylation

of E2 thereby inactivating it

PUROMYCIN - an analogue of tyrosinyl-tRNA

which causes premature release of polypeptide

- is nonselective and acts on both 70s and 80s

ribosomes

CYCLOHEXAMIDE - acts only on eukaryotic cellsby inhibiting Peptidyltransferase on 60s ribosomes


60/65

VIRUSES AND PROTEIN SYNTHESIS

Contain either DNA (pox, herpes) or RNA (HIV, Rabies) butnot both

Some can direct the host cell to make DNA from its RNAtemplate via REVERSE TRANSCRIPTASE - retro/oncoviruses

Uses host cell processes to replicate their nucleic acid andsynthesize viral coat proteins - all viruses

May compete with host cell for limited translation factors

may also inhibit host mRNA from binding to 40s ribosomeby dephosphorylation of BP1

May disrupt 4G-4E complex so that viral translationbecomes more efficient than host translation -picorna/poliovirus

S S S G N C MAC N O


61/65

VIRUSES USE GENETIC MACHINERY OF

HOST


62/65


63/65

INTERFERONS

Proteins that are synthesized by virus-infected cells

Viral double-stranded nucleic acid stimulates theexpression of the interferon gene

Interferon is carried to other cells to prevent viralreplication and stop further infection

All viruses are capable of eliciting interferon production

Interferon for a specific virus is active against many

other different viruses Mechanism of action : triggers synthesis of ANTIVIRAL

PROTEIN


64/65


65/65

HAVE A GREAT WEEK!

And God

said, Let us

make man in

our own

image, afterour likeness

Genesis 1:26

Date post:	09-Apr-2018
Category:	Documents
Upload:	len-arellano
View:	214 times
Download:	0 times

Translation doc tengco

Documents