Slide 1 : PROTEIN SYNTHESIS - II
ADDITIONAL INFORMATIONS
15-10-2011 5.30 PM By
Dr. Ichha Purak
http://www.dripurak.com http://www.drichhapurak.webnode.com 1 Appendix 10/14/2011
Slide 2 : AN INTRODUCTION TO AMINO ACIDS
PEPTIDE BOND
RIBOSOME
GENETIC CODE
TRANSCRIPTION
MESSENGER RNA
TRANSFER RNA 2 Appendix 10/14/2011
Slide 3 : A BRIEF INTRODUCTION ABOUT AMINO ACID
Amino Acids (20) are monomers (building blocks ) of proteins.
General formula for amino Acid is RCHNH2COOH,
Because of having both basic (Amino-NH2) and acidic (Carboxylic - COOH ) group, amino acids are amphoteric.
The side chain (R) or alkyle radical of Amino acids vary and may possess additional amino or carboxylic group which may develop charges. These additional charges play an important role in determining 3-D structure of proteins and formation of active sites in proteins acting as enzymes.
The side chain of amino acids may be simple or complex, may contain hydroxyl, acidic,basic ,sulphur or aromatic group. 3 Appendix 10/14/2011
Slide 4 : AMINO ACID STRUCTURE 4 Appendix 10/14/2011
Slide 5 : TYPES OF AMINO ACIDS ON NATURE OF SIDE CHAINS 5 Appendix 10/14/2011
Slide 6 : 20 Protein Amino Acids and their 3 letter abbrebiation in Genetic code 2 Non standard Amino Acids
Selenocysteine encoded by UGA in Archaea & Eubacteria
Pyrrolysine encoded by UAG in Archaea & Bacteria . 6 Appendix 10/14/2011
Slide 7 : AMINO ACID BIOSYNTHESIS
Amino acids are synthesized by using Carbon skeleton from intermediates of TCA cycle, Glycolysis, Calvin Cycle,Pentose phosphate Pathway etc. The intermediates like 3PGA,Oxalo Acetic Acid, Keto Glutaric Acid, Pyruvic acid, PEPA,Shikimic Acid etc.Which take up Amino group from glutamic acid by Aminotransferases . For synthesis of Glutamic Acid , Ammonia is fixed by symbiotic (Rhizobium leguminosarum) and Non symbiotic ( Azotobacter and Clostridium) micro organisms. 7 Appendix 10/14/2011
Slide 8 : Two Amino Acids are linked by peptide linkage, liberating a molecule of water. During peptide bond formation carboxylic acid group of one Amino Acid reacts with amino group of other Amino Acid to form amide group.
This is a dehydration synthesis reaction (also known as a condensation reaction).The resulting O=C-NH bond is called a peptide bond, and the resulting molecule is an dipeptide PEPTIDE BOND FORMATION
R1CHNH2COOH +HNHR2CHCOOH →R1CHNH2C=ONHR2CHCOOH + H2O Peptide unit is rigid and planar.Carbon –Nitrogen linkage is a partially double bond. It is not free to rotate. The length of this bond is 1.32Aᵒ which is between that of a single bond C-N(1.49Aᵒ ) and a double bond C=N(1.27Aᵒ ) The other single bond surrounding peptide bond is flexible and is not rigid O=C-N-H 8 Appendix 10/14/2011
Slide 9 : PEPTIDE BOND FORMATION Peptidyle Transferase 9 Appendix 10/14/2011
Slide 10 : RIBOSOME Ribosomes are Ribonucleoprotein non membranous particles .
Ribosomes are the site of Protein Synthesis
Ribosomes are present in cytoplasm either free or attached to ER or nuclear membrane.
In eukaryotic cells ribosomes are also present in mitochondria and plastids.
Prokaryotic ribosomes are 70S particles having 2 subunits 30s & 50s
Eukaryotic cytoplasmic ribosomes are 80S particles having 2 subunits 40S & 60 S
The two sub units are dissociated at low Mg++ concentrations and associate at high Mg++ concentrations
The smaller subunit fits on larger subunit leaving a tunnel between two,which can accommodate the mRNA , aminoacyl tRNAs and other factors during protein synthesis.
Both the sub unit of ribosome contain rRNAs and proteins 10 Appendix 10/14/2011
Slide 11 : Ribosomes are dumb bell shaped or spherical having a diameter of 140-160Aᵒ
Number of ribosomes/cell is directly related to RNA content and protein synthesis rates.
Many ribosomes can bind same messenger RNA one after one for polypeptide synthesis of same nature forming polysome
Ribosomal RNA are large molecules ,larger than diameter of ribosome,remains coiled like a spring with helical and non helical regions.
rRNA of smaller unit helps in correc t orientation of mRNA on ribosome during protein synthesis
2 ribosomal units after organisation in nucleolous pass to cytosol and aggregate to form complete functional ribosomal complex in cytosol. 11 Appendix 10/14/2011
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Slide 15 : GENETIC CODE
It has been possible to design the features of genetic code by the contributions made by Nirenberg, Khorana and Holley (1968) who got the Nobel Prize for Medicine for their work of synthesis of polypeptide using artificially synthesized mRNA.
The Genetic code is a dictionary that identifies
the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual word is composed of three nucleotide bases. These genetic words are called codons.The assembledge of codons is like a sentence. This sentence keeps the information for deciding sequence of Amino acids in a polypeptide 15 Appendix 10/14/2011
Slide 16 : The information of DNA is decoded into polypeptide chain through formation of mRNA by transcription
mRNA carries message of DNA as Genetic code
Genetic code is the sequence of deoxyribonucleotides ( A T C G ) present on the fragment of DNA ,which acts as Gene and is transferred onto mRNA as sequence of ribonucleotides ( A U C G )
Genetic code is combination of codons . Each codon is sequence of 3 bases ( Triplet ) which specifies a Amino Acid
As there are 4 bases (A T C G ) ,a combination of 3 bases can give 64 codons ( 4x4x4 =6 4 ) which are more than adequate for 20 protein Amino Acids. Some AAs have more than one codon. 16 Appendix 10/14/2011
Slide 17 : Properties of Genetic code
The code is triplet( 3 bases/letters )
Code is universal A particular codon specifies a particu;ar Amino Acid in all organisms
Code is degenerate – more than one codon for a Amino acid
In case of more than one codon for a amino acid 1st 2 bases
are specific and 3rd is flexible or wobble .One tRNA can recognise all 4 codons Leucine – 4 codons CUU,CUC,CUA,CUG
Code is comma less having no punctuation 17 Appendix 10/14/2011
Slide 18 : Code is non over lapping One base is used (read ) only once
Code in non ambiguous , codes for same amino acid ,whether present at the beginning or intercalary Exception GUG Met (start) Val (Intercalary)
Out of 64 codons 61 are assigned to different Amino Acids
3 codons UAA,UAG and UGA donot specify any Amino acid act as Termination codons
AUG & GUG are the 2 Initiator codons .3rd UUG Archaebacteria
Leu, Arg & Ser-6 codons, Val,Pro,Thr,Ala & Gly-4 codons
Met & Trp – one codon
Colinearity exists between DNA, mRNA & polypeptide chain 18 Appendix 10/14/2011
Slide 19 : Genetic Code As RNA Codons
Leu , ser & Arg -6 codons
Val, Pro,Thr,Ala & Gly -4 codons 19 Appendix 10/14/2011
Slide 20 : GENETIC CODE AS DNA CODONS T C A G T C A G T
C
A
G
T
C
A
G
T
C
A
G
T
C
A
G 20 Appendix 10/14/2011
Slide 21 : Transcription is the synthesis of RNA from a DNA template. Whether prokaryotic or eukaryotic, has three main events.
Initiation - binding of RNA polymerase to double-stranded DNA; this step involves a transition to single-strandedness in the region of binding; RNA polymerase binds at a sequence of DNA called the promoter.
Elongation - the covalent addition of nucleotides to the 3' end of the growing polynucleotide chain; this involves the development of a short stretch of DNA that is transiently single-stranded
Termination - the recognition of the transcription termination sequence and the release of RNA polymerase TRANSCRIPTION 21 Appendix 10/14/2011
Slide 22 : Although transcription is performed by RNA Polymerase, the enzyme needs other proteins to produce the transcript. These factors are either associated directly with RNA Polymerase or add in building the actual transcription apparatus. The general term for these associated proteins is transcription factor Promoter - all the DNA sequences containing binding sites for RNA polymerase and the transcription factors necessary for normal transcription 22 Appendix 10/14/2011
Slide 23 : Transcription involves synthesis of RNAs(mRNA,tRNA,rRNA) on DNA template, resembling on of the two DNA strands (Sense strand )
Transcription is carried out by RNA Polymerase enzyme and a number of factors.
In Prokaryotes there is only one RNA Polymerase responsible for synthesis of all the three RNAs and is assisted by Sigma (ᵟ) factor
RNA Polymerase Recognises the Initiation site located near Promoter by sigma factor.
Local unwinding of DNA double helix takes place by dissolving H bonds between base pairs
One of the two separated strands act as template on which RP advances and start recruiting complementary ribonucleotide tri phosphates 23 Appendix 10/14/2011
Slide 24 : RNA synthesis generally starts with ATP or GTP and all the 3 phosphates are retained with first nucleotide. Once elongation starts sigma factor is released . Transcription continues till stop signal is provided by Rho factor or hair pin loop formation due to presence of palindromic sequence.
Prokaryotic promoter site is present near the transcription Initiation site and contains Pribnow box (TATAAT) to the left (10 bp) of Initiation site and a second consensus sequence (TTGACA) about 35 bp upstream
In Eukaryotes there are 3 different RNA Polymerase enzymes for synthesis 3 RNAs. RP are assisted by number of transcription factors. 24 Appendix 10/14/2011
Slide 25 : RP I transcribes rRNA, RP II mRNA and RP III transcribes tRNA. The eukaryotic promoter for mRNA has TATA box ( 25-31 bp upstream) and CAAT box (70-80 bp upstream) to Initiation of Transcription site 25 Appendix 10/14/2011
Slide 26 : A Transcription unit includes a promoter, an RNA coding region and a terminator 26 Appendix 10/14/2011
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Slide 29 : MESENGER RNA (mRNA) mRNA comprises only about 5% of the total RNA in the cell, but is most heterogenous type of RNA in the cell. Coding region varies greatly.
The mRNA carries genetic information from the DNA to cytosol, where it is used as a template for protein synthesis.
mRNA is short lived between several seconds to few minutes in bacteria but longer lived in eukaryotes possibly because it has to move out of nucleus to the site of protein synthesis in the cytoplasm
In Prokaryotic organisms ribosome can attach to mRNA,while it is being transcribed. In this situation translation begins at the 5’ end of mRNA while the 3’ end is still attached to DNA 29 Appendix 10/14/2011
Slide 30 : mRNA molecule may contain 1200-1500 or mor bases in its coding region and can code for more than 500 AAs.
Only a small amount of DNA is used for coding mRNA
The mRNA is single stranded and contains genetic code in its coding region in the form of sequence of codons (Specific for a particular Amino Acid) ,Each codon is triplet of bases.
The message of mRNA is read consecutively in 5’→3’ direction. Genetic code is Universal,Comma less,non overlapping ,degerate and Non ambiguous.
Prokaryotic mRNA undergoes very little processing after trancription. In prokaryotes transcription and translation are coupled.
Precursor Eukaryotic mRNA possess both coding regions (Exons) and Non coding (Introns) regions and require some processing to give mature function form. 30 Appendix 10/14/2011
Slide 31 : General features of Eukaryotic mRNA are as follows
A cap of ATP/GTP is added at the 5’ end after transcription.G or A get methylated for it. Cap facilitates binding of mRNA with ribosome and provide stability
cap is followed by Non coding (UTR) region I ,which is used for binding of mRNA with smaller sub unit through its single rRNA.
Initiator codon follows AUG which directs to introduce Met at first position.
It is followed by coding region (1200-1500 nucleotides) and is furnished with genetic code
Towards 3’ end one of the three termination codon provides signal for termination of Polypeptide chain. 31 Appendix 10/14/2011
Slide 32 : mRNA may be monocistronic ( coding for one Polypeptide ) or polycistronic (coding for more than one polypeptide) In case of polycistronic mRNAs there may be 2nd noncoding region followed by 2nd coding region .
on the 3’end A poly Adenine ( 50-150 ) tail is added after transcription which also provides stability to mRNA
In case where introns are present , By spilcing introns are removed and exons in sequence are ligated to form continuous stretch of coding region. 32 Appendix 10/14/2011
Slide 33 : Simple illustration of an unspliced mRNA precursor, with two introns and three exons (top). After the introns have been removed via splicing, the mature mRNA sequence is ready for translation (bottom) 33 Appendix 10/14/2011
Slide 34 : Transcription & Translation 34 Appendix 10/14/2011
Slide 35 : Features of Eukaryotic mRNA Features of Prokaryotic mRNA 35 Appendix 10/14/2011
Slide 36 : TRANSFER RNA (tRNA ) or Soluble RNA Transfer RNA is a small RNA chain (73-95 nucleotides ) that transfers a specific amino acid to a growing polypeptide chain at ribosome during translation.
It acts as a adapter molecule because it can recognise both a specific Amino Acid as well as its codon on mRNA. For loading 20 different protein Amino Acids there are different tRNAs.(1-20)
It acquires a clover shaped three dimensional structure due to having many unusual or minor bases
It is single stranded but is folded on itself and has some helical region and some loops.
All tRNAs share some common features : 36 Appendix 10/14/2011
Slide 37 : tRNAs are single stranded having 73-95 ribonucleotides
Many unusual bases are present. Some bases are methylated
5’ end generally has Guanine or Cytosine
Base sequence at 3’end is CCA. The activated Amino Acid is attached to 3’OH group of ribose of terminal Adenosine
About half nucleotides in tRNA molecule form helical structures by complementary base pairing forming 4 arms
Group of unpaired bases form a loop T ψC loop at the end of T 5 bp arm
The anticodon arm is a 5-bp stem whose loop contains the Anticodon
The Anticodon loop has 7 bases with following sequence
5’ Py-Py –X-Y-Z- Modified Pu- variable bases 3’. X-Y-Z act as Anticodon and is variable
There is an extra or variable arm 37 Appendix 10/14/2011
Slide 38 : A 4 bp D arm ends in DHU loop ,to this loop amino acyl synthetase enzyme binds.This loop has several dihydrouracil residues
tRNAs have following Recognition sites
Amino Acid attachment site at 3’end CCA terminity
Ribosome Recognition site (T ψC ) loop
The Anticodon site 3 middle unpaired bases X-Y-Z form Anticodon . By presence of Anticodon tRNA recognises complmentary codon on mRNA and is also able to pick up specific Amino Acid
Enzyme Recognition site ( DHU loop )
Variable loop or Extra Arm ( Its function is yet not known ) 38 Appendix 10/14/2011
Slide 39 : Features of tRNA 39 Appendix 10/14/2011
Slide 40 : THE END 40 Appendix 10/14/2011