From Gene to Protein

Chapter 17 : From Gene to Protein Over View • • Genetic material : -is the information content of DNA Or – it is specific sequences of nucleotides along strands of the DNA . The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins . -Proteins are the links between genotype and phenotype (this is the main point of this chapter). • Concept 17.1 Gene expression: the process by which DNA directs protein synthesis, includes two stages: Transcription and Translation : Genes specify proteins via transcription and Translation. -How was the fundamental relationship between genes and proteins discovered? • Evidence from the Study of Metabolic Defects  In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell . So , genes dictate the production of a specific enzyme , and the inability to synthesize a certain enzyme leads to inherited disease.  Biochemists accumulated much evidence that, cells synthesize and degrade molecules via metabolic pathways , in which each chemical reaction is catalyzed by a specific enzyme .( such metabolic pathway leads to synthesis of pigments that give the color of eyes of fruit fly )  Nutritional Mutants in Neurospora - Scientific Inquiry - George Beadle and Edward Tatum exposed bread mold called (neurospora crassa )to X-rays, for creating mutants (they wanted mutants which are unable to survive in minimal medium . Note : in minimal medium wild-type bread mold can survive ; by their metabolic pathways they produce all the other molecules they need , while mutants couldn’t because they were unable to synthesize certain essential molecules from the minimal ingredients , so they need COMPLETE MEDIUM or minimal medium with additional nutrients , to grow . 1|Page what did Beadle & Edward do ? - they want to charecterize the metabolic defect in each mutant , they took samples from grown mutant and distributed them in vials (vials contained minimal mediums+single additional nutrient ) ; the particular additional nutrient that allowed the growth indicated the metabolic defect . - for example if the suplemented vial that supported growth of the mutant was the one contain amino acid (argenine) they will conclude that mutant was defictive in synthesise of (argenine) . Note : Argenine biosynthesis involved precursor nutrient + intermediate molecules (ornithine & citruline ) together. And that needs many enzymes . their expierment below is about (Do individual genes specifiy the enzymes that function in abiochemical pathway) - mutant cell cannot grow because it needs areginine (it cant produce it ) - mutants that required argenine in their growth are fell in 3 classes , each lacking a different necessary enzyme for synthesizing argenine . -they grew each one of these 3 classes into 4 conditions to know what the differences between these 3 classes of mutants . the results are in the figure >> (MM) means (minimal medium) 1st column : wild type was capable to growth in all conditions , requiring only the minimal medium . 1st row : no nutrient was added to the minimal medium (it's pure precursor nutrient) so mutant cell wasn’t grown in any one . 4th row : argenine was added directly so the mutant cell was grown in the 3 classes . -the three classes of mutants each had a specific s growth requirements , for example (class ll) mutants couldn’t grow when ornithine alone but could grow when either citrulline or argenine was added . 2|Page Conclusion : - Each class of mutant was unable to carry out one step in the pathway for synthesizing argenine , because it lacked the necessary enzyme which catalyze that blocked step. Steps of argenine synthesis : 1.synthesis (ornithine) needs (precursor nutrients) and (enzyme A) 2. synthesis (Citrulline) needs (Ornithine) and (enzyme B) . 3. synthesis (Argenine ) needs (Citrulline) and (enzyme C). In another word >> No enzyme A >it needs a ready ornithine to begin the synthesis way of argenine … From this inquiry >> - they concluded that each mutated gene must normally dictate the production of one enzyme . -They developed a( one gene–one enzyme hypothesis), which says that the function of the gene is to dictate production of a specific enzyme . • But , researchers later revised the hypothesis: one gene–one protein because Some proteins aren’t enzymes and Many proteins are composed of several polypeptides, each one has its own gene . Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis , but it is common to refer to gene products as proteins rather than polypeptides  3|Page • Basic Principles of Transcription and Translation - - Genes provide instructions for making specific proteins but not directly , the bridge between DNA and Protein is the nucleic acid RNA . • Transcription is the synthesis of RNA under the direction of DNA Both nucleic acids (DNA & RNA) use the same language , and information is simply transcribed or copied from one molecule to another . 1. DNA molecule serves as a template for assembling a complementary sequence of RNA nucleotides . 2. Then the RNA molecule will carry the genetic massage from the DNA to Protein synthesizing machinery of the cell . This type of RNA called Messenger RNA (mRNA) . Note transcription is the general term for the synthesis of any kind of RNA on a DNA template , not only for mRNA synthesizing . -------------------• - Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA During this stag there is a change in language : the cell must translate the base sequence of an mRNA molecule into the amino acid sequence of a polypeptide . the sites of translation are Ribosomes ( complex particles that facilitate the orderly linking of amino acids into polypeptide )  Transcription and Translation occur in all organisms , the basic mechanics are similar for bacteria and eukaryotes , but there is an difference in the flow of genetic information within the cell. -In prokaryotes, mRNA produced by transcription is immediately translated without additional processing . - 4|Page Because of lacking for nuclei ; DNA is not segregated (separated) from ribosomes that allows translation of an mRNA to begin while its transcription is still in progress. -in eukaryotes , the nuclear envelope separate transcription from translation in space and time . Transcription occur in nucleus and mRNA is transported to the cytoplasm where translation occurs. -before the mRNA leave nucleus , it modified in various way to produce the final functional mRNA . 1. The primary transcript : The initial RNA transcript from any eukaryotic gene ( in protein coding) which results in Pre-mRNA. 2. Pre-mRNA will modified in various ways to produce the final functional mRNA. • We concludes that , cells are governed by a cellular chain of command: DNA → RNA → protein (this concept was dubbed The central dogma ). Revision from chapter 5 : What are the differences between DNA & RNA ? RNA contains ribose while DNA contains deoxyribose RNA has the nitrogenous base uracil , while DNA has nitrogenous base Thymine. RNA molecule consists usually of a single strand . • The Genetic Code • 5|Page When biologists guessed that the instructions for protein synthesis were encoded in DNA they recognized a problem : There are 20 types of amino acids , but there are only four types of nucleotide bases in DNA ! How many bases correspond to an amino acid?! 1- The flow of information from gene to protein is based on a triplet code a series of (non-overlapping, three-nucleotide )words . triplet code = the genetic instructions which are written in the DNA for the polypeptide chain. • These triplets are the smallest units of uniform length that can code for all the amino acid . Example: this triplet code (AGT) at a particular position on a DNA strand results the amino acid (serine) at the corresponding position of the polypeptide chain . Figure17.4 : The Triplet Code 1)During Transcription, one of the two DNA strands called the template strand works as a template for ordering the sequence of nucleotides in an RNA transcript. Note the RNA molecule is synthesized in an anti-parallel direction to the template strand of DNA. 2) During Translation, the mRNA base triplets, called codons, they are translated (decoded) by translation machinery in the 5′ to 3′ direction ,each codon specifies the addition of one of 20 amino acids . Notes : 1. mRNA molecule is complementary ,not identical to its DNA template  2.the term codon is also used for the DNA base triplets along non-template strand , and thus identical in sequence to the mRNA except they have (T) instead of (U) .for this reason the non-template DNA strand called the coding strand ;) 3. the number of nucleotides in mRNA must be 3 times more than the number of the resulted amino acids . (12 nucleotide on mRNA = 4 amino acids ) 6|Page  Cracking the Code • All 64 codons were deciphered by the mid-1960s . look to figure below   Of the 64 triplets, 61 code for amino acids ; and 3 triplets are “stop codons” signals to end translation (UAA , UAG , UGA).  the codon( AUG) has a dual function > 1. it codes for the amino acids methenine (Met) 2. functions as a "start " signal or initiation codon . So , genetic messages must begin with the mRNA codon (AUG) which signals the protein –synthesizing machinery to begin translating mRNA . And because this codon stands for the amino acid ( methonine) , polypeptide chains also must begin with (methonine) . however, an enzyme may then remove this starter amino acid from the chain . • The genetic code is redundant but not ambiguous ; no codon specifies more than one amino acid(no ambiguity) , but more than one codons may specify the same amino acid (redundancy) Example : the amino acid (Pro)is specified by 4 codons . look to the figure • Codons must be read in the correct reading frame (correct groupings) in order producing a specified polypeptide. ** Although a genetic message is written with no space between the codons , the cells protein- synthesizing machinery reads the message as a series of non overlapping three letters words (the message isn’t read in this way UGGUUU >> it is read in this way : UGGUUUGGC ) . 7|Page  Evolution of the Genetic Code - The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals . there are exceptions to this universality ( as example there are a slight variations *in the genetic code exist in certain unicellular eukaryotes . and in* translating stop codons into one of two amino acids not found in all organisms ) but it’s a small variations! - Genes can be (transcribed and translated after being transplanted) from one species to another. Example : bacteria can be programmed by the insertion of human genes to synthesize certain human proteins for medical use , such as Insulin. Concept Check ^_^ Q1 : what polypeptide product would you expect from a poly-G mRNA that is 30 nucleotides long ? you can use the last table  Q2 : the template strand of the gene contains the sequence (3‘-TTCAGTCGT-5‘) , a) drawn 1 – the non-template sequence 2- the mRNA sequence indicating 3 and 5 ends of each .. b) imagine that the non template sequence is transcribed instead of the template sequence , draw the mRNA sequence and translate it . you can use the last table. Answers : Q1 : the poly peptide made up of 10 Gly (glycine) amino acid . Q2 : a) 1- non template sequence : 5‘-AAGTCAGCA-3‘ 2- mRNA sequence : 5‘-AAGUCAGCA-3 ‘ b) 1- you have to write the non template sequence again conversely (3‘-5‘) before transcription non template sequence template sequence 5‘-AAGTCAGCA-3‘ 3‘-ACGACTGAA-5‘ 2- mRNA sequence = 5‘-UGCUGACUU-3‘ 3- Translated polypeptide = Cys-STOP- Leu (from the table in page 7) 8|Page Concept 17.2 : Transcription is the DNA-directed synthesis of RNA (a closer look) Transcription, the first stage of gene expression, can be examined in more details >> a) Molecular Components of Transcription : An enzyme called an RNA Polymerase pries the two strands of DNA apart and joins the RNA nucleotides as they base-pair along the DNA template. - This enzyme (like the DNA polymerase that functions in DNA replication ) can assemble the nucleotides only in its (5‘ 3‘) direction , and (unlike DNA polymerase ) it is able to start a chain from scratch ; it doesn’t need a primer . - RNA synthesis follows the same base-pairing rules as DNA, except (Uracil ) substitutes for (Thymine) and the type of nucleotides. - Specific sequence of nucleotides along the DNA mark where transcription of a gene begins and ends : • Promoter : The DNA sequence where RNA polymerase attaches . • Terminator : the sequence signaling the end of transcription . - biologists refer to the direction of transcription as "downstream" and the other direction as "upstream" , these terms are also used to describe the positions of nucleotide sequences within DNA and RNA. So the promoter sequence is up – stream in DNA from the terminator . - The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit .  Figure 17.7 : the stages of Transcription Initiation : after RNA polymerase binds to the promoter , The DNA strands unwind , and the polymerase initiates RNA synthesis at the start point on the template strand . Elongation : The polymerase moves downstream , unwinding the DNA and elongating the RNA transcript (5 3). in the wake of transcription , the DNA strands re-form a double helix . Termination : eventually , the RNA transcript is released , and the polymerase detaches from the DNA. 9|Page Note while bacteria has a single type of RNA polymerase that synthesize not only mRNA but also another types of RNA , eukaryotes have at least 3 types of RNA polymerase in their nuclei , the one that used for mRNA synthesis is called (RNA polymerase II) . b) Synthesis of an RNA Transcript consists of 3 stages : – Initiation – Elongation – Termination - – stage 1 RNA Polymerase Binding and Initiation of Transcription Promoter’s roles are : serving as a binding site for RNA polymerase , determining the initiation of RNA synthesis and determining which one of the two strands of the DNA helix is used as a template . • In bacteria RNA polymerase itself recognizes the promoter and binds to it, but in eukaryotes there is a collection of proteins called "Transcription factors" mediate the binding of RNA polymerase and the initiation of transcription (only after certain transcription factors are attached to the promoter RNA polymerase II bind to it) . **in eukaryotes** - transcription factors and RNA polymerase II biund to a promoter to form a completed assembly called a" Transcription initiation complex" . - a section of promoter (a promoter DNA sequence) called "TATA box" is crucial in forming the initiation complex at the eukaryotic promoter . (without it transcription factors will not bind to DNA promoter and so polymerase will not binds too ! ) Figure 17.8 : the initiation of transcription at look to next figure >> eukaryotic promoter Note : the interaction between eukaryotic polymerase II and the transcription factors is an example of the importance of protein – protein interactions in controlling eukaryotic transcription. Once the polymerase is firmly attached to the promoter DNA , the two DNA strands unwind there and the enzyme starts transcribing the template strand. 10 | P a g e Stage2 : Elongation of the RNA Strand - RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time. -Transcription progresses at a rate of 40 nucleotides per second in eukaryotes - A gene can be transcribed simultaneously by several RNA polymerases following each other, Which helps the cell make the encoded protein in large amounts . 1.the enzyme adds nucleotides to the 3‘ end of the growing RNA molecule . 2.in the wake of RNA synthesis , The new RNA molecule peels away from its DNA template and the DNA double helix reform . 11 | P a g e Stage 3 : Termination of Transcription • The mechanisms of termination are different in bacteria and eukaryotes - In bacteria, the polymerase stops transcription at the end of the terminator ( polymerase detach from the DNA and release the transcript , which is available for use as mRNA) - In eukaryotes, 1. RNA polymerase II transcribes a sequence on the DNA called the polyadenylation signal sequence , which codes for a polyadenylation signal (AAUAAA) in the pre-mRNA .. 2. at a point about 10 to 35 nucleotides downstream from the AAUAAA signal , the proteins cut it free from the polymerase forming (premRNA) , but polymerase continues transcribing . in another word : in eukaryotes , (polymerase doesn’t release from DNA template at the same time with pre-mRNA transcript ).  (recent research suggests that the RNA produced by the continued transcription (after pre-mRNA released) is digested by enzyme that moves along the RNA ; when this enzyme reach the polymerase , transcription is terminated and the polymerase falls off the DNA . 12 | P a g e Concept 17.3 : • Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm - During RNA processing, both ends of the primary transcript are usually altered. Also, usually some interior parts of the RNA molecule are cut out, and the other parts spliced together . These modifications produce an mRNA molecule ready for translation … • Alteration of mRNA Ends - Each end of a pre-mRNA molecule is modified in a particular way: – The 5′ end receives a modified nucleotide (5′ cap) – The 3′ end gets a poly-A tail   5′ cap = a modified form of (Guanine) nucleotide added onto the 5′ end , after transcription of the first 20 to 40 nucleotides . (not translated into protein) Poly A-tail = (50 – 250) (Adenine) nucleotides added to the 3′ end . (not translated into protein) - These modifications share several functions: - They seem to facilitate and promote the export of mRNA from nucleus . They protect mRNA from degradation from hydrolytic enzymes . They help ribosomes attach to the 5′ end ( in conjunction with cytoplasmic proteins ) when mRNA arrive to cytoplasm . Look to the figure >> Figure 17.9 : RNA processing : addition of the 5 cap and poly-A tail UTR (Un Translated Region): parts of the mRNA that will not be translated into protein , but they have other functions such as ribosome binding . 13 | P a g e • Split Genes and RNA Splicing  -  Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions .that means the sequence of DNA nucleotides that codes for a eukaryotic polypeptide is usually not continuous , its split into segments . These non-coding regions are called intervening sequences, or introns The other regions are called exons because they are eventually expressed ( usually translated into amino acid sequences ). But , there is an exception; UTR codes of the exons at the ends of the RNA , which make up part of the mRNA aren’t translated into protein !! because of this exceptions ,you can say Exons are "sequences of RNA that exit the nucleous " , with this definition UTR are part of Exons  RNA splicing removes introns and joins exons, this process creating an mRNA molecule with a continuous coding sequence . Note : the term exons and introns are used for both RNA sequence and the DNA sequence that encodes them . Look to next figure Figure : 17.10 "RNA processing : RNA Splicing"  figure (17.10) shows RNA molecule codes for B-globin .. - The number under the RNA refers to codons , in this figure > it consists of 164 codon . - There are 3 exons , those will form the sequence of mRNA .. - During RNA processing the introns will cut out and exons spliced together. 14 | P a g e How is Pre-mRNA splicing carried out ? Researchers have concluded that >> 1) There is a short nucleotide sequence at each end of an intron works as a signal for RNA splicing. 2) particles called small nuclear RiboNucleoProteins (snRNP) recognize these splice sites. 3) Several different snRNPs join with additional proteins to form Spliceosome (which is almost as big as Ribosome) . Splicesome interacts with certain sites along an intron , releasing the intron and joining together the two exons .. small nuclear RiboNucleoProteins (snRNP) pronounced "snurps" -they are located in the cell nucleus -they are composed of RNA and protein molecules **RNA in those particles called small nuclear RNA . Its Role : - Catalyze the previous process. - Participate in spliceosome . - Recognize Splice sites . Figure 17.11 : The Role of snRNP and spliceosomes in pre-mRNA splicing. Forming Splicesome on a pre-mRNA molecule Within the splicesome snRNA base-pairs with nucleotides at a specific sites of along the intron. Splicesome releases the intron and at the same time splices the exons together .. and the result mRNA contains exons only 15 | P a g e <> • Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA, that splicing can be called self-splicing. • For example in Protozoan Tetrahymena self splicing occurs in the production of ribosomal RNA (rRNA) "a component of the organisms ribosomes". The pre rRNA actually removes its own introns . • The discovery of ribozymes disagreed the belief that all biological catalysts were proteins • Three properties of RNA enable it to function as an enzyme (aribozymes) 1- It can form a three-dimensional structure because of its ability to base pair with itself . 2- Some bases in RNA contain functional groups . 3- RNA may hydrogen-bond with other nucleic acid molecules . Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide  Molecular Components of Translation • the cell keeps its cytoplasm stocked with all 20 amino acids in the (cytoplasmic pool of amino acids ) ; by synthesizing them from other compounds or by taking them up from the surrounding solution . • A cell translates an mRNA message into protein in ribosomes with the help of transfer RNA "tRNA" . - The mRNA message is a series of codons. tRNA molecules transfer the amino acids from cytoplasm to a ribosome. Ribosomes adds each amino acid brought to it by tRNA to the growing end of a polypeptide chain (join the amino acids into a chain) Note : Molecules of tRNA are not all identical : - Each tRNA molecule translates a particular mRNA codon into a particular amino acid , how ? Each tRNA carries a specific amino acid on one end . and has an anticodon (nucleotide triplet )on the other end ; the anticodon base-pairs with a complementary codon on mRNA (by hydrogen bonds). 16 | P a g e look to figure 17.13 " Translation :the basic concept" This figure shows that >> 1) As the mRNA is moved through a ribosome , codons are translated into amino acids , one by one . 2) tRNA adds its amino acid to a growing polypeptide chain when the anticodon hydrogen bonds to a complementary codon on the mRNA . Q : tRNA is a translator , how ? because it can read a nucleic acid word (mRNA codon) and interpret it as a protein word (the amino acid ) . 17 | P a g e a) The Structure and Function of Transfer RNA • • • • • tRNA Like other types of cellular RNA ; it is transcribed from DNA templates . and in eukaryotic cells its made in nucleus like mRNA and then travel to the cytoplasm ,where translation occurs . tRNA in both bacterial and eukaryotic cells is used repeatedly ; after transferring an amino acid to ribosome goes to cytoplasm and pick another one and transfer it , and so on .. A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long (it is shorter than mRNA) tRNA , this single strand can fold back upon itself and form a molecule with a threedimensional structure by hydrogen bonds between its bases . when tRNA is Flattened into one plane it looks like a clover leaf, and when it is twisted and folds in 3-D structure it is roughly has L-shape . Figure 17.14 The structure of transfer RNA (tRNA) a) In Two dimensional structure : - In all tRNA , there are 4 base-paired regions with 3 loops , and base sequence of the amino acid attachment site at the 3‘ end . - The colored sequences in these 3 loops are unique to each tRNA type. so anti-codon is unique too. b) In three dimensional structure : -The loop extending from one end of the (L -shaped tRNA) includes the anti-codon . - the other end of (L-shaped tRNA) protrudes its (3‘ end) which is the attachment site for amino acid. c) look to anti-codon its written as 3‘ to 5‘ sequence for aligning properly with codon which is 5‘ to 3‘ sequence . 18 | P a g e  Accurate translation requires two processes of molecular recognition :  - First : a correct match between a tRNA and an amino acid, done by a family of related enzymes called aminoacyl-tRNA synthetases The active site of each type of aminoacyl-tRNA synthetase fits only a specific combination of amino acid and tRNA . there are 20 different synthetases . Synthetase catalyses the covalent attachment of the amino acid to its tRNA in a process driven by the hydrolysis of ATP .and the resulted aminoacyl tRNA also called charged tRNA which is ready to go ribosome. - 1. Active site binds the amino acid and ATP. 2. ATP losses 2 phosphategroups and joins amino acids as AMP. 3. appropriate tRNA covalently bonds to amino acid, displacing AMP . 4. The tRNA charged with amino acid is released by the enzyme.  - Second: a correct match between the tRNA anticodon and an mRNA codon There are 61 types of mRNA codons while only 45 types of tRNA anti-codons .. because of that , some of tRNA s must bind to more than one codon . For example : the base U at the 5-end of the anti-codon can bind with A or G in the third position(at the 3-end) of an mRNA codon ... Flexible pairing at the third base of a codon is called wobble Wobble explains why one amino acid can be presented by more than one codon differs in their third bases only  19 | P a g e b) Ribosomes • Ribosomes facilitate specific coupling of tRNA anti-codons with mRNA codons in protein synthesis - The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA "rRNA" (rRNA form 2/3 of ribosome mass , and it’s the most abundant type of RNA in the cell ) - In eukaryotes the subunits are made in the nucleolus . when the rRNA is transcribed , it will be processed and assembled in nucleus with imported protein from cytoplasm to form the subunits , which will exported to cytoplasm via nuclear pores. - In bacteria and eukaryotes , large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule . Picture (a) : When the polypeptide becomes longer it passes through "exit tunnel" in the large subunit , and when its become complete it leaves to cytosole. picture (b) & (c) • A ribosome has three binding sites for tRNA: – The P site : holds the tRNA that carries the growing polypeptide chain – The A site : holds the tRNA that carries the next amino acid to be added to the chain – The E site : it is the exit site, where discharged tRNAs leave the ribosome. • After addition the new amino acid to the carboxyl end of the growing polypeptide . ribosome catalyze the formation of peptide bond . Note : Recent researches support the hypothesis that the rRNA is responsible for both structure and function of ribosome ; it’s the main constituent of the interface between the 2 subunits and( A and P sites) and it’s the catalyst of peptide bond formation . What about protein ? it supports the shape changes of the rRNA molecules during translation. 20 | P a g e  Building a Polypeptide - we can divide translation (synthesis of polypeptide ) into three stages : – Initiation – Elongation – Termination - All three stages require proteins “factors” that aid the translation process - energy is provided by the hydrolysis of GTP( Guanosine Triphosphate) . - The polypeptide is synthesized always in one direction from N-terminus (initial amino acid end ,met) toward the C-terminus (carboxyl end ). a) Ribosome Association and Initiation of Translation The initiation stage of translation brings together mRNA, tRNA with the first amino acid, and the two ribosomal subunits : figure(17.17)  1. the small ribosomal subunit binds with mRNA and a special initiator tRNA which carried the amino acid (methionine) and anti-codon (UAC). 2. the small subunit moves along the mRNA until it reaches the start codon (AUG) . - In bacteria : small subunit binds to mRNA at a specific nucleotide sequence in eukaryotes : small subunit with the initiator tRNA are already bound and then bind to the 5‘cap of the mRNA (which is upstream to the start codon) . then they move downstream along the mRNA until they reach the start codon , and tRNA hydrogen bonds to it . - Start codon : it signals the start of translation ; it establishes the codon reading frame for the mRNA . 3. The union of mRNA & initiator tRNA& small ribosomal subunit is followed by attachment of large ribosomal subunit and forming Translation Initiation Complex . and that requires : - Proteins called initiation factors are required to bring all these components together . - GTP molecule is required to form the initiation complex .  21 | P a g e When the initiation process is completed , the initiator tRNA sits in the P site of the ribosome and A site will be ready for the next aminoacyl tRNA . Figure 17.17 The initiation of translation b) Elongation of the Polypeptide Chain - During the elongation stage, amino acids are added one by one to the preceding amino acid Each addition involves proteins (elongation factors) and occurs in three steps cycle : codon recognition, peptide bond formation, and translocation. Energy consuming occurs in the first and third steps . each one requires hydrolysis of one GTP . This cycle takes less than a tenth of second (0.1 sec)in bacteria , and is repeated at each amino acid is added to the chain until the polypeptide is completed. Figure 17.18 The elongation cycle of translation 1 2 3 22 | P a g e Figure(17.18) Illustration : 1.codon recognition : the anticodon of an incoming aminoacyl tRNA base-pairs with complementary mRNA codon in the A site . hydrolysis of the GTP increases the accuracy and efficiency of this step . 2. peptide bond formation : an rRNA molecule of the large ribosomal subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site .. this step removes the polypeptide from the tRNA in the P site and attaches it to the amino acid on the tRNA in the A site . 3. Translocation : The ribosome translocates the tRNA in the A site to the P site . and the empty tRNA in the P site is moved to the E site , where its released . the mRNA moves along with its bound tRNAs , bringing the next codon to be translated into the A site . -mRNA moves in the ribosome in one direction only ( from 5‘ end ) . and it moves relatively to each other (unidirectionally) , codon by codon . c) Termination of Translation - - Termination occurs when a stop codon (UAG, UAA,UGA) in the mRNA reaches the A site of the ribosome . a protein called a release factor (shaped like tRNA) binds directly to the stop codon in A site, and that causes the addition of a water molecule instead of an amino acid to the poly peptide chain . This reaction breaks (hydrolyzes)the bond between the completed polypeptide and the tRNA in the P site. releasing the polypeptide through the exit tunnel . translation assembly then comes apart (dissociate) in a multistep process , aided by other protein factors and requires hydrolysis of 2 more GTP molecules . Figure 17.19 The termination of translation 23 | P a g e  Polyribosomes • A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) • Polyribosomes enable a cell to make many copies of a polypeptide very quickly . • Poly ribosome are found in bacteria and eukaryotes . Figure 17.20 Polyribosomes  Completing and Targeting the Functional Protein -Often translation is not sufficient to make a functional protein ,Polypeptide chains are modified after translation and Completed proteins are targeted to specific sites in the cell . 24 | P a g e a) Protein Folding and Post-Translational Modifications  During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape (secondary or tertiary structure) . Thus , the gene determines the primary structure , and primary structure in turn determines shape. ** in many cases , a chaperone protein helps the polypeptide fold correctly .  Proteins may also require post-translational modifications before doing their job, for example : 1- Certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups or other addition . 2- Enzymes may remove one or more amino acids from the leading (amino) end of the polypeptide chain . or cleaved polypeptide chain into 2 or more pieces . 3- 2 or more polypeptides that are subunits of a protein that has quaternary structure , a familiar example is hemoglobin. b) Targeting Polypeptides to Specific Locations  Two populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and bound ribosomes (attached to the ER) , both they are identical and can switch their status from free to bound . - Remember : free ribosomes synthesize proteins that stays in the cytosol and function in it. While bound ribosomes synthesize protein of the endo-membrane system and proteins secreted from the cell .  Polypeptide synthesis always begins in the cytosol , and finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER  Polypeptides destined for the ER or for secretion are marked by a sequence of amino acids(about 20 amino acid)near the leading (amino) end called signal peptide - a protein- RNA complex called signal-recognition particle (SRP) binds to the signal peptide . it brings the signal peptide and its ribosome to the ER (to receptor protein built into ER membrane this receptor is part of a multi-protein translocation complex). Figure 17.21 The signal mechanism for targeting proteins to the ER 4 1 5 6 3 2 25 | P a g e Figure 17.21 Illustration : 1- polypeptide synthesis begins on a free ribosome in the cytosol. 2-An SRP binds to the signal peptide , halting synthesis momentarily . 3-The SRP bind to a receptor protein in the ER membrane . This receptor is part of a protein complex ( a translocation complex) that has a membrane pore and a signal-cleaving enzyme. 4-The SRP leaves , and polypeptide synthesis resumes , with simultaneous translocation across the membrane (The signal peptide stays attached to the translocation complex) . 5-the signal-cleaving enzyme cuts off the signal peptide . 6-the rest of the completed polypeptide leaves the ribosome and folds into its final conformation . if it’s for secretion it will released within the ER lumen , and if it is membrane protein it remains partially embedded in the ER membrane . Note 1 : There are another Kinds of signal peptide that target the protein to organelles which aren’t of endo-membrane system . and in these cases translation will be completed in cytosol before the polypeptide is embedded in the organelle . Note 2 : bacteria also employ signal peptides to target proteins for secretion . Concept check : 1. what two processes ensure that the correct amino acid is added to a growing polypeptide chain ? first , each aminoacyl-tRNA synthetase specifically recognize a single amino acid and attaches it only to an appropriate tRNA . second, tRNA charged with its specific amino acid binds only to an mRNA codon for that amino acid . 26 | P a g e Concept 17.5: Point mutations can affect protein structure and function • Mutations are changes in the genetic material of a cell or virus • Point mutations are chemical changes in just one base pair of a gene - The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein Point mutation may lead to many disorders , such as heart condition that are responsible for sudden death in young athletes and Sickle –cell disease .. Figure 17.22 The molecular basis of sickle-cell disease: a point mutation  Types of Point Mutations  Point mutations within a gene can be divided into two general categories 1- Base-pair substitutions 2- Base-pair insertions or deletions 1- Base-pair substitutions  base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides .  depending on its effect , we can categorize it to : look to figure 17.23 - Silent mutations : have no effect on the amino acid produced by a codon because of redundancy in the genetic code (change in codon but no change in amino acid) - Missense mutations (most common substitution mutation) still code for an amino acid, but not necessarily the right amino acid and it has a little effect on the protein ; the amino acid may have properties similar to those of the amino acid it replaces , or the new amino acid is in a non essential region to protein function . 27 | P a g e - Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein because the resulting polypeptide will be shorter than the polypeptide encoded by the normal gene . Figure 17.23 Types of point mutations Original 28 | P a g e 2- Insertions and Deletions - Insertions and deletions are additions or losses of nucleotide pairs in a gene - These mutations have a disastrous effect on the resulting protein more often than substitutions do - Insertion or deletion of nucleotides may alter the reading frame, producing a frame shift mutation (when the number of nucleotides inserted or deleted isn’t multiple or three) . Figure 17.23 Types of point mutations - All the nucleotides downstream of the deletion or insertion will be improperly grouped into codons , and the result will be , extensive missense and usually ending sooner or later in nonsense . - Unless the frame shift is very near the end of the gene , the protein is almost certain to be nonfunctional . 29 | P a g e original  Mutagens - We studied that , When there is an error in replication it will be corrected by a system that we learned about in chapter #16 . otherwise the incorrect base will be used as a template in the next round of replication , resulting in a mutations . - The rate of mutation is about (1 nucleotide in every 1010 is altered ). - Mutagens are physical or chemical agents that can cause mutations , such as : Physical mutagens > X-rays , ultraviolet light (which can cause disruptive Thymine diamers in DNA ). Chemical mutagens > they affect the structure of DNA and they are carcinogenic >> examples of types -Base analogs : Chemical mutagens that are similar to normal DNA bases , but they pair incorrectly during DNA replication . - some Chemical mutagens insert themselves into the DNA and distorting the double helix . - Concept check : What happens when one nucleotide pair is lost from the middle of the coding sequence of a gene ? The answer is in (insertion and deletion) part  - What if a gene whose template strand contains the sequence (3’-TACTTGTCCGATATC-5’) is mutated to (3’-TACTTGTCCAATATC-5’) For both normal and mutant genes draw 1- the double stranded DNA 2- resulting mRNA 3- amino-acid sequence ( depend on the table page 7). Normal Mutant 1- double DNA strand 3‘- TACTTGTCCGATATC-5’ 5’-ATGAACAGGCTATAG-3‘ 3‘-TACTTGTCCGATATC -5’ 5’-ATGAACAGGTTATAG-3‘ 2- mRNA sequence 5’-AUGAACAGGCUAUAG-3‘ 5’- AUGAACAGGUUAUAG -3‘ 3- amino acid sequence Met-Asn-Arg-Leu-stop Met-Asn-Arg-Leu-stop - 30 | P a g e The mutation has no effect  because the amino acid sequence still as it was .. Concept 17.6: While gene expression differs among the domains of life, the concept of a gene is universal - before 40 years organisms were divided into 3 categories : bacteria , archea, eukaryotes. - Archaea are prokaryotes, but share many features of gene expression with eukaryotes  Comparing Gene Expression in Bacteria, Archaea, and Eukarya - Bacteria and eukarya differ in their RNA polymerases, termination of transcription ; archaea tend to resemble eukarya in these respects . - bacterial and eukaryotic differ in ribosomes and initiation of translation, archea ribosome are the same size with bacterial ribosome , but their sensitivity to chemical inhibitors is most closely to eukaryotic ribosomes , and in initiation it is more like bacterial process . - Bacteria ( with absence of nucleus) can simultaneously transcribe and translate the same gene . and the resulted protein quickly diffuse to its site of function . In eukarya, nuclear envelope separates transcription and translation . it provides a compartment of extensive RNA processing that leads to have additional steps in the process In archaea, since it lack nuclear envelope ,transcription and translation are likely coupled . - - Figure 17.24 Coupled transcription and translation in bacteria The Idea of this figure >> In bacterial cells the translation of mRNA can begin as soon as the leading (5‘) end of mRNA molecule peels away from the DNA template . 31 | P a g e  What Is a Gene? Revisiting the Question - The idea of the gene itself is a unifying concept of life • We have considered a gene as: - A discrete unit of inheritance - A region of specific nucleotide sequence along the length of a DNA molecule in a chromosome (chapter 16) - A DNA sequence that codes for a specific polypeptide chain (chapter 17) Figure 17.25 A summary of transcription and translation in a eukaryotic cell The 2 subunits dissociate after each translation . Finally we can define gene as >> a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule . 32 | P a g e Quick Summary : types of RNA >> 33 | P a g e