Lecture 10 - Protein Synthesis & Ribosomes PDF

Lecture 10 bacterial ribosomes two subunits - large and small measure in units of S the large subunit is 50 S, and small is 30 S = 70 S which doesn't make sense and this is because the S scale is not linear, but it...

Lecture 10 bacterial ribosomes two subunits - large and small measure in units of S the large subunit is 50 S, and small is 30 S = 70 S which doesn't make sense and this is because the S scale is not linear, but it is related to mass under centrifugal force eukaryotes 60 + 40 = 80S what are ribosomes composed of rRNA proteins - these are structural and do not catalyse any reactions large S small S in the prokaryotic there is a 23 S eukaryotic ribosomes Lecture 10 1  large is 60 S small is 40 S together is 80 S ribosomal RNA is 28 S 23 S and 28S catalyse reaction that the ribosome factory does i.e. the formation of a peptide bond between the amino acids to generate a polypeptide catalytic activity in ribosome function which is therefore acting in an enzymatic capacity, making it a ribozyme if you use proteases that degrade most proteins of the ribosome you will still get a catalytic activity, whereas if you use RNAse to degrade the RNA in the ribosome, you destroy the catalytic activity 16 S, and 18 S recognition in the signal in the genome as towards translating of the protein 5S in both structural 5,8 S is extra in eukaryotes involved in translocation the genetic code DNA is written as a triplet code - codon Lecture 10 2  each of these position you could have any of the four bases, so you have a possibility of 64 codons a double code would not give you enough to specify the 20 proteinogenic amino acids if we have a quadruple code is excessive so triplet is great of those 64 codons,how many specify amino acids 61 specify, degenrate but there are only 20 proteinogenic amino acids which means some amino acids will be encoded by more than one codon (degeneracy) alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutamine Gln Lecture 10 3  Q glutamic acid Glu E glucine Gly G histidine His H isoleucine Ile I leucine Leu L lycine Lys K methionine Met M Phenylalanine Phe F proline Lecture 10 4  Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr Y valine Val V ribosome does what reads mRNA, read in reading frames meaning of reading frames reading in triplets depends on where you start, meaning you get different reading frames this is important because the wrong codon being read will mean the wrong amino acid is encoded - this however does not generally happen because there are stop signals fairly soon afterwards to stop the amino acid from being transcribed Lecture 10 5  types of point mutations missense mutation nonsense mutation frameshift mutation missense mutation substitution of one nucleotide so that an alternative amino acid is incorporated into the polypeptide nonsense mutation substitution of one nucleotide so that a stop codon is prematurely introduced, and this results in early termination of the polypeptide frameshift mutation a point mutation which results in the ribosome either reading four nucleotides as three, or backing up one nucleotide and reading from a different reading frame methionine is specified by AUG, start codon the code is Lecture 10 6  universal but codon usage varies among different organisms universality allows us to take a code and put it into a gene, e.g. insulin and put it into a bacterial host ribosomes read that code and produce insulin that is how insulin is produced distinctive codons of human mitochondria ribosomes protein synthesising factories include mRNA which carries the genetic info in the form of codons tRNAs are adaptors between codons and amino acids - keys to deciphering codons rRNA associates with a set of proteins to form ribosomes reaction between amino acids inside the ribosome join amino acid of one tRNA to the next Lecture 10 7  the first amino acid (next to chain) will undergo a nucleophilic attack from the other one, breaking a bond between the tRNA and the first amino acid this leads to the second amino acid to form a bond between the tRNA and it the ribosome will translocate along so that everything moves so that the second tRNA moves along where are the new amino acids attached to C terminus of the growing polypeptide chain how do ribosomes exist exist in the cell as separate units of large and small subunits only together during translation 1) the small subunit will bind to mRNA Lecture 10 8  2) the initiator tRNA is the key between the code and for the correct anticodon, it will have the correct amino acid at the 3' end bound to the small subunit and the messenger RNA 3) the large subunit joins to form a complete ribosome and so translocation happens 4) when it encounters a stop signal, the polypeptide is released, small and large units separate and the whole thing can start over again can there be multiple ribosomes translating the same mRNA yes they are called polyribosomes can happen in prokaryotes and eukaryotes but eukaryotes cannot do cotranscriptional translation) because there is a nucleus so transcription can happen in the nucleus tRNA Lecture 10 9  tRNAs single stranded molecule of RNA are adaptors between codons and amino acids is encoded by hundreds of tRNA genes organised in tandem arrays tRNA is transcribed by RNA Pol III undergoes some processing - you can get some unusual bases occurring (highlighted) why does tRNA have a loops Lecture 10 10  region of complementarity creating loops that are characteristic of clover leaf shape for tRNA structure of tRNA attached amino acid T loop D loop anticodon - complementary sequence to codon on mRNA two-step decoding processes in tRNA in the 3' end, addition of CCA group, where amino acid (cognate amino acid) added there is a dedicated enzyme (amino acyl tRNA synthetase) for adding each amino acid to the tRNA e.g. for tryptophan there is tryptophanyl tRNA synthetase this process is very accurate utilising energy of ATP, there is a high energy bond established at the 3' end to add the amino acid on the 3' end the amino acid is correctly selected by the codon (as it is already charged by the time it comes to bind to the mRNA) why is it so accurate? because the ribosome itself can't determine which amino acid is present what is an amino-acyl tRNA tRNA with an amino acid at the 3' end tRNA can tolerate not quite correct pairing between codon and antiocodon if there are 61 codons, there would be 61 matching tRNAs, HOWEVER, most cells do not have 61 tRNAs humans have around 48-50, number varies between species Lecture 10 11  how do few tRNAs recognise all these codons? some tRNAs can recognise multiple codons how do tRNAs recognise codons wobble phenomenon at the wobble position, at the 3rd base at the mRNA, and 1st base at the tRNA if you have any of these bases (C A G U, in blue), they will do their standard pairing, they will also do some non-standard pairing inosine is a derivative of adenosine - mostly found in tRNA Lecture 10 12  why does wobble phenomenon happen tRNA is a single stranded molecule anticodon loop - you can see that the bases on this part are much closer together between 33-34 there is a big (5' end) = first position of anticodon this big gap allows toleration of mismatches how does inosine arise adenosine loses its amine group inosine will pair with U and C Lecture 10 13  it changes the directionality of hydrogen but maintains the number of H bonds and it will pair with other bases due to the spacing in tRNA, even adenosine and inosine can occur (even though they are both purines) shine-dalgarno sequence AUG is not a random codon cell needs to known WHICH AUG does this through shine-dalgarno sequence - in prokaryotes (different in eukaryotes), allows the interaction of the small subunit of the ribosomal RNA, recognises the shine-d sequence, binds to it and enables correct positioning over AUG consensus sequence - most commonly occurring (COULD differ but generally this one) fMet in prokaryotes how does cell know it is fMET there are initiator tRNAs which are the only ones that can bind here Lecture 10 14