this chapter covers gene expression or how the information carried on a gene is used to make a protein we will first have an overview of a gene expression we will then move on to the first step of gene expression which is transcription then to the genetic code and translation in a second video part two translation on the er of the endoplasmic reticulum will be covered and finally a last video part 3 will cover mutations let's start with overview of gene expression how are genes related to dna genes are segments of dna that code for a particular protein or rna molecule think of dna being a recipe book and each gene being a recipe to make a protein the human genome contains about 3 billion base pair and about 25 000 genes most genes encode proteins but some genes products are rna molecules that play a variety of role in cells as we will see later on the expression of most gene involves two distinct processes transcription of a gene into rna a quick reminder that rna uses u instead of t transcription creates a copy of a gene it occurs in the nucleus of an eukaryotic cell translation is the production of a protein using the information carried by the rna transcript formed during transcription this is where ribosomes play an important role it occurs in the cytoplasm i like to compare gene expression to the co to cooking you have a recipe book your dna with many recipes from barbecue sauce to chocolate cake a friend wants to make the chocolate cake you brought to them you will not give them your book they don't care about the barbecue sauce so you make a copy of a chocolate cake recipe this will be the rna your friend the ribosome will use the instruction on the copy of the recipe v rna to make the chocolate cake the protein notice in this analogy that the recipe book is made of paper just like the copy of a recipe dna and rna are both made of nucleotide but the chocolate cake is the actual food item it is not paper a protein is not made of nucleotide it is not a nucleotide a nucleic acid keep this picture in mind the same sequence transcription and translation is found in all cells but bacterial cells lacking a nucleus have transcription and translation both happening in the cytoplasm in contrast the two steps are happening in different locations in eukaryotic cells transcription occurs in the nucleus the primary rna will then be processed this processing does not occur in bacterial cells the process rna exits the nucleus and enters the cytoplasm where it will be translated into a protein the template strand of dna is used to make a complementary strand of rna during transcription the rna is then read and the corresponding amino acids are added to form a protein book copy cake as a reminder a quick reminder before we move on rna is single stranded its nucleotides contain a ribose the bases rna nucleotides carry could either be a for adenine c for cytosine g for guanine or u for uracil dna is double stranded its nucleotide contain a dioxyribose the base dna nucleotides carry could either be a for adenine c for cytosine g for guanine or t for thymine transcription the stages of transcription are initiation elongation and termination during initiation an enzyme called rna polymerase binds to a special region called promoter the two dna strands separate during elongation the rna polymerase copies of a dna template strand to make a copy in the form of a complementary rna strand the rna gets longer in termination the rna polymerase reaches the end of the gene and stop transcribing the rna transcript is released the initiation of transcription starts at an eukaryotic promoter eukaryotic promoters contain a short sequence called a tata box its name comes from the sequence of bases notice it is written a's and t's and remember that there are two hydrogen bonds linking this bases this makes it easier to break than g and c which have three bonds that means it is easier to separate the two dna strand in regions rich with a and t's rather than g's and c's transcription factors are molecules that assist rna polymerase in binding to the promoter and starting transcription during elongation the two strands of dna are separated rna polymerase in gray reads the template strand of dna shown in the dark blue and makes a copy in the form of a complementary rna strand by adding complementary nucleotide for each t on the dna template strand rna polymerase adds an a on the rna strand for each a it adds a u remember this is rna we are making for each g it adds a c another important thing to notice rna polymerase reads the dna strand from 3 prime to 5 prime and makes an rna strand from 5 prime to 3 prime remember the direction of nucleic synthesis that has been covered in the previous chapter in bacteria two processes can lead to termination in which the rna polymerase reaches the end of a gene and stops transcribing the rna transcript is released this could be done either through self-termination in this process the transcription of sequences within the terminator region causes rna to fold into a stem-loop structure causing the loosening of a grip of rna polymerase on the dna everything is released termination could also be enzyme-dependent rho which is a termination enzyme pushes through between rna polymerase and dna releasing the polymerase in eukaryotic cells the transcriber rna needs to be processed we call this rna the primary rna not that this happens in eukaryotic cell not in bacteria the primary rna transcript will receive a five prime cap on its five prime side and a poly a tail on its free prime side entrance will be spliced out we will come back to what our entrance in the next slide look at the picture below the yellow region represents the five prime cap and the polyethylene this has been added to the primary rna transcript the red region shows the region that has been transcribed by rna polymerase so transcribed by rna polymerase and this has been added and this has been added notice the start codon and the stop codon the region in between is the coding protein coding segment this is the region containing the information to make a protein the region before the start codon is called the utr or let's write it five prime utr is a five prime and trans lei tied region u t r the region after the stop codon is called the three prime utr over 3 prime untranslated region the illustration below shows an rna with its 5 prime cap and 3 prime polyetail it also shows exon in red and introns in pitch the rna below has been spliced that means its introns have been removed and the exons bound together so to recapitulate the introns are regions that are removed whereas exons are kept and will be part of a coding region which will be used to make a protein how is splicing carried out by structures called spliceosome they are made of protein and small nuclear rna or snrna fn rna contain a region complementary to the end of the entrance sequence the base pairing the entrance is splice out or removed the exons are joined a genus sequence may contain many introns and exons for example brca1 a gene involved in dna repair and those variants are often linked to breast cancer has 24 exons exons tend to code for distinct substructures called domains in protein for example in this picture exon 1 corresponds to domain 1 exon 2 correspond to domain 2 and exon 3 correspond to domain 3. exon shuffling is a molecular mechanism resulting in the formation of new genes in this process two or more exons from different genes are brought together the new reage arrangement creates a gene with altered function axon shuffling is thought to be an evolutionary process by which new genes are made but be careful exon shuffling is different from splicing occurs during the process of a primary rna where he has exon shuffling occurs in at the level of dna and can create new genes rna transcript can have various rules they can be messenger rna or mrna a mrna is a copy of a gene that encodes a protein but for some genes the end product is the rna itself this rna will not be translated into a protein example of such rna are ribosomal rna or rrna that are structural component of ribosome and transfer rna or trna which deliver the correct amino acids to ribosome during the synthesis of proteins before we move on to the next stage of gene expression we need to go over the genetic code the basis of a genetic code is the correspondence of nucleotide sequence in the rna and amino acids in proteins each amino acid in a protein is specified by sequences of three nucleotides these sequences are called codons each of the 20 amino acid is coded for by a unique set of codons for example rna sequence e u g correspond to the amino acid mesionine this codon is particularly important since it signals the start of translation and these ends called the start codon the rna sequence ggn n being any of the four bases so n could be a u c or g meaning it could be ggg ggc gga or ggu so ggn corresponds to the amino acid glycine and c-a-a or c-a-g both correspond to the amino acid glutamine as you can see in this example more than one codon can code for the same amino acid glycine has four well there is only 20 amino acid and 64 possible codons there has to be some redundancy in the genetic code which is a very good thing as we will see later the correspondence of these 64 codons with amino acid is orange in the genetic code table let's practice we have a dna sequence presented here remember the conversion is to have a 5 prime and on the upper left side the top strand being the sand strand the bottom being the anti-sense strand of a template dna this is the strand read by rna polymerase remember it reads from 3 prime to 5 prime and it makes from 5 prime to 3 prime so the template is copied into the complementary rna strand during transcription the rna strand will be used to make a protein another thing to remember is that the mrna is a bit longer than the coding sequence go back to the processing of rna that means the first thing to do is to look for the start codon a u g once found we can start making a protein for this we need to split the rna sequence in threes or codons then we look at the table e u g the start codon code for methionine you let's look for fifiu in the table here it is it codes for pro short for proline so we have met and pro the next is g g g here it is it codes for the amino acid glycine gly for short the next is si e e this code for gln or glutamine finally we have u e g this sequence along with u a a and u a g indicates the end of translation they are stop codon it is not an amino acid i write stop in bracket but really i don't even need to write it i can just stop rgln note that since the start codon is aug all proteins begin with met the genetic code is universal the expression of genes in all living organisms uses the same genetic code this is why we can take a gene from a firefly and introduce it into a plant the plant will make the corresponding protein luciferase and it will shine in the same manner a gene from a jellyfish can be introduced into a pig the pig cell will express the gene into its protein another luciferase by the way the big nose and feet shine so these are just two examples but i could cite many more the message is that we can express gene from an insect into a plant or a gene from a mollusk into a mammal the next stage of gene expression is translation translation the basic concept the main player of translations are ribosome shown in brown in this drawing they facilitate the production of polypeptide by first matching codons found in the mrna with a complementary anticodon in trna shown in light green and then catalyzing the peptide bonds between amino acid shown in purple carried by trna on the left we see a depiction of a ribosome and its part on the right a ribosome at work that means reading a mrna and interacting with a proper trna to synthesize a protein let's remember that a ribosome is formed by two subunits a large and a small one the small subunit contains the binding site for the coming mrna the large subunit contains a three site the a site receives the incoming trna carrying an amino acid the p site all the trna that carries the growing polypeptide the e site will be the exiting site for the mtrna notice the name of these sites are linked to their function aside for amino acid trna binding site think of the trna carrying the single amino acid p site for peptidyl trna binding site or growing polypeptide or protein p protein and e site for exit transfer rna or trna for short is a molecule of rna folded in a specific manner two regions are of importance the anticodon site which is composed of three nucleotides that base pair with a complementary codon in mrna that means if the mrna has a codon a g c then the trna codon will be ucg the trna shown here has the anticodon a-a-g-a-a-g this means its mrna corresponding codon will be u u c u u c codes for phenylalanine or p phae this means this trna will carry the amino acid phenylalanine on that a second region of importance which is the amino acid attachment site an amino acid rna synthesis is an enzyme that joins a specific amino acid to its corresponding trna energy is required in this process and is provided by atp translation has three phases initiation elongation and termination translation initiates when the small ribosomal subunit aligns with the start codon a aug of mrna the initiator trna with the anticodon u a c complementary to the start codon aug comes in it carries methionine the large subunit then joins the complex during the elongation cycle of translation a ribosome moves along the mrna reading its codons and allowing corresponding trna to join and bring their amino acid amino acids are then added to the growing polypeptide one by one a trna is already on the p side carrying the growing polypeptide let's call this trna trna 1 a new trna with an anticodon matching the mrna newly exposed codon on the l side comes in let's call it trna two tyrenet2 is now sitting right next to trna1 this allows the polypeptide bond to form between the newly brought amino acid and the previous amino acid the growing polypeptide chain moves from trna1 to trna2 the ribosome keeps sliding along the mrna the empty grna1 is now found on the e side it leaves the ribosome trnnet2 is now on the p side and the cycle goes on a new trna so we have now two here and here we're going to have a third trna that comes in and carrying the new polypeptide and etc etc translation ends when a stop codon is reached a stop codon could be either uag uaa or uga there is no corresponding trna for this codons instead a release factor fits in the air side and catalyzes the dissociation of all components the mrna is freed the two ribosomal subunits separate and the protein is released let's imagine we are ribosome first thing first the small subunit needs to find the start codon e u g b the small subunit and the mrna come in contact a trna with the anticodon u a c comes next it carries the mechanin amino acid the large sum unit completes the ribosome placing the into the p site so we'll have a s i t and we'll have e site here make sure to know the direction of the mrna the five prime on the left and the three prime on the right the ribosome is going to move into that direction a new trna with the anticodon matching a c e comes next and position itself on the a site so it has the anticodon u g the amino acid it brings it let's look at the table and find a c a the codon is sea so if we look at the table we're going to find a c a and it's called fofreonin that means we're going to have a freonin amino acid carried a peptide bond is then created between these two amino acids the growing polypeptide made of two amino acids moves on onto the second tyranny the ribosome slides towards the three prime end so let's draw a ribosome with its site so the second trna is now on the p side the first crna emptied is on the e side it exit so let's get rid of it it's done it's gone the next codon on the a side is a u g that means the anticodon is going to be u e c so that's the next trna that comes in yes aug is the start codon but it mephenine can be part of a protein it is not just for the start codon so we are going to have mephionin next a peptide bond is formed the ribosome moves along so let's the ribosome moves along so the newly emptied trna reaches exit and leaves the ribosome so this one has rich it exits it's gone the next codon shown is u g a which is a stop codon a release factor comes in everything comes apart the protein is released the small subunit goes the large subunit goes refactor the trna and that's it we now have a protein made of three amino acid polyribosomes are a formation of multiple ribosomes translating the same mrna at once this means several proteins can be made from one mrna like several cakes can be made from one copy of a recipe chaperonins are large protein structure that assist newly made polypeptides to ensure they fold properly they capture and properly folded protein inside its cavity forcing them to unfold and hopefully we fold correctly as mentioned previously some ribosomes are three in the cytoplasm these ribosomes produce protein needed within the cytoplasm overs are found attached to the ear why this ribosome in fact can synthesize proteins that will not be used in the cytoplasm but that could be necessary in the lumen of the er or in lysosome or destined to be secreted we will come back to that later how is this possible because of the presence of a signal peptide in the n-terminus of a protein being made and these targets this growing protein to the er lumen this process will be described in more details in the next video thank you