2 DNA replication? Review What proteins are involved? DNA polumerase? Heelicase?Primer?Ligase?
3 Transcription The synthesis of mRNA Transcription produces mRNA. RNA is SINGLE stranded. Transcription can take place on either of the exposed strands. The template strand = ANTISENSE STRAND. The other is the SENSE STRAND.
4 Transcription is always carried out in a 5' - 3' direction So the 5’ end of the free RNA nucleotide is added to the 3’ end of the RNA molecule that is already synthesized.
5 Have a look at these…Theory follows these slides….
6 DNA consists of two polynucleotide strands, only one of which is transcribed into RNA The antisense strand is transcribed into RNA. This is the TEMPLATE STRAND.The sense strand is not transcribed into RNA. It has the SAME BASE SEQUENCE as the mRNA.Can identify the antisense strand because mRNA must be transcribed in the direction 5’ 3’
7 Also on page 202 (old book) page 117 new book. Average length of an mRNA molecule in mammals is 2000 nucleotides in length.Again, you do not need to draw this from memory but you do need to be able to explain what is going on!Also on page 202 (old book) page 117 new book.
8 Remember that…A gene is a sequence of DNA which is transcribed into RNA and contain three main parts:Promoter: Responsible for the initiation of transcription (in prokaryotes, a number of genes may be regulated by a single promoter - this is an operon)Coding Sequence: The sequence of DNA that is actually transcribed (may contain introns in eukaryotes)Terminator: Sequence that serves to terminate transcription (mechanism of termination differs between prokaryotes and eukaryotes)The coding sequence is divided into sections, each section is called a codon. One codon is made of three bases aka a base triplet.
9 In SummaryTranscription is the process by which a DNA sequence (gene) is copied into a complementary RNA sequence and involves a number of steps:RNA polymerase binds to the promoter and causes the unwinding and separation of the DNA strands2. Nucleoside triphosphates (NTPs) bind to their complementary bases on the antisense strand (uracil pairs with adenine, cytosine pairs with guanine)3. RNA polymerase covalently binds the NTPs together in a reaction that involves the release of two phosphates to gain the required energy4. RNA polymerase synthesises an RNA strand in a 5' - 3' direction until it reaches the terminator5. At the terminator, RNA polymerase and the newly formed RNA strand both detach from the antisense template, and the DNA rewinds6. Many RNA polymerase enzymes can transcribe a DNA sequence sequentially, producing a large number of transcripts7. Post-transcriptional modification is necessary in eukaryotes. (Introns must be removed to form mature mRNA.)
10 The synthesis of polypeptides on ribosomes. TranslationThe synthesis of polypeptides on ribosomes.DNA RNA Protein (poly peptide)Table 1, p.g. 119 lists all 64 codons. There are only 20 amino acids, so more than one codon is able to code for each a aminio acid. This is why the code is sometimes described as ‘degenerate’ because there is more than one codon for each amino acid (e.g. GUU, GUC = valine = stop codon).
11 Ribosomes Viewed with an electron microscope Large subunit + small subunitSubunits composed of ribosomal RNA (rRNA) as well as many proteinsRibosomes formed in nucleolus of eukaryotic cells. Exit nucleus through pores.Translation occurs between the subunits.Figure 9, p.g. 118.
12 What do the sites do?Site A – holds tRNA carrying next amino acid to be added to the chainSite P – holds tRNA carrying growing polypeptide chainSite E – tRNA lost amino acid, ready to be discharged.You do not have to be able to draw the structure of a ribosome. But you MUST know the order of the sites.
13 There are four stages of translation First some important back ground info.Codon AUG on the 5’ end of all mRNA. Start codon.Hydrogen bonds form in four places of the tRNA strand – forms the ‘clover leaf structure’ (fig pg 205).One of the loops has an exposed anticodon, this is unique to the tRNA and pairs with the mRNA.There are 20 amino acids which can bond to the tRNA. Bonding occurs due to enzymes. How many enzymes are there?Active site on enzymes allows specific amino acids to bond to specific tRNA. To make the attachment, ATP must supply energy. The structure is now called an activated amino acid. tRNA ready to deliver amino acid.You do not have to be able to draw the clover leaf diagram but you should be able to recognize it and explain why this shape is achieved.
14 Initiation The process starting An activated amino acid (methionine) attached to a tRNA with the anitcodon UAC. Combines with a small ribosomal subunit and an mRNA strand.Subunits moves down mRNA strand until start codon is reached (AUG)Hydrogen bonds form between the initiator tRNA and the start codon.A large ribosomal subunit combines as well, producing a translation initiation complex.Initiation factors are proteins which join the complex together.Energy in this case does not come from ATP. It comes from Guanosine triphosphate (GTP) which is another energy rich molecule – very similar to ATP.
15 2. Elongation the chain growing tRNA’s bring amino acids to the mRNA-ribosomal complex. Order specified by the codons.Proteins called elongation factors assists binding tRNA’s to the mRNA codons at site A.Initiator tRNA then moves to site P.Ribosomes catalyse the formation of peptide bonds between amino acids.
16 3. Translocation Takes place during elongation tRNA relocating within the ribosomeTakes place during elongationtRNA moving from site A P E.Occurs in a 5’ to 3’ direction so ribosomal complex is moving along the mRNA strand towards the 3’ end.
17 4. Termination There are three stop codons. The process endingThere are three stop codons.When one reaches site A, a release factor (protein) fills site A.The release factor does not carry amino acid. It causes the bond linking the the tRNA to the P site to hydrolyse. Releasing the polypeptide from the ribosome.The ribosome then separates form the mRNA and splits into it’s subunits.Most proteins produced this way are used in the cell. However is a protein is produced by a ribosome bound to the endoplasmic reticulum (ER) the protein will be secreted from the cell or used in the lysosomes.