Biology 1001 Unit 9: DNA to Protein PDF

Biology 1001 SECTION 9 From Gene to Protein All information for this section can be found in CHAPTER 17 *BEGIN reading at BASIC PRINCIPLES OF TRANSCRIPTION AND TRANSLATION (p 359-364) 17.2 Transcription is the DNA-directed synthesis of RNA: A closer look (p 364- 367) – ONLY the fir...

Biology 1001 SECTION 9 From Gene to Protein All information for this section can be found in CHAPTER 17 *BEGIN reading at BASIC PRINCIPLES OF TRANSCRIPTION AND TRANSLATION (p 359-364) 17.2 Transcription is the DNA-directed synthesis of RNA: A closer look (p 364- 367) – ONLY the first section (Molecular components of transcription) (p 364-5) 17.3 Eukaryotic cells modify RNA after transcription (p 367-369) 17.4 Translation is the RNA-directed synthesis of a polypeptide: A closer look (p 369- 378) Possible Exam Figures Figure 17.4 Overview: the roles of transcription and translation in the flow of genetic information. (p 361) Figure 17.5 The triplet code. (p 362) Figure 17.15 Translation: the basic concept. (p 370) Figure 17.17 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA. (p 371) Figure 17.25 A summary of transcription and translation in a eukaryotic cell. (p 379)  We already know that genes control an organism’s phenotypic traits : ◼ The colour of Mendel’s pea flowers, the white eyes of Thomas Hunt Morgan’s mutant fruit flies, the white hair of the Kermode bear,  i.e. what it looks like, and its physiology  But how do genes determine an organism’s characteristics?  What does a gene actually say???  Concept 17.1 – Genes specify proteins via transcription and translation Current knowledge of how GENES work:  Genes are sequences of DNA that contain the instructions for making proteins  The instructions are not used directly but copied out on RNA molecules  Messenger RNA carries the instructions to the protein- making machinery in the cytoplasm of the cell  Proteins are produced  And PROTEINS control the traits (characteristics) of living organisms  PROTEINS are the link between our DNA and our CHARACTERISTICS  DNA dictates the synthesis of proteins, via intermediary RNA molecules  And look what proteins do! In 1956, Francis Crick summarized the cellular flow of genetic information in his CENTRAL DOGMA  Crick said that  Cells are governed by a molecular chain of command  DNA RNA Protein  Proteins run all the metabolic processes of life  This is still almost 100% true GENE EXPRESSION culminates in the production of PROTEINS  Gene Expression proceeds in two stages  1. TRANSCRIPTION – imagine that you transcribe one book into another, using in different fonts but the same language (nucleic acid letters)  Using a DNA template to produce RNA script  2. TRANSLATION – you translate into another language (nucleic acid into a sequence of aminoacids)  Using mRNA (messenger RNA) as an instruction to produce a polypeptide  NOTE: In some cases, we stop at p.1 - the end product of transcription is rRNA (ribosomal RNA) or tRNA (transfer RNA).  These are used as is, (i.e. not translated into a protein product), so in these cases, gene expression stops at transcription  Basic Mechanisms of Transcription and Translation are Similar, but Not Identical in Bacteria and Eukarya  Bacteria  Eukarya  No nuclear membrane – DNA in the  Nuclear membrane separates nucleoid region transcription and translation in space and time  Translation can begin while transcription is still in progress  And mRNA is processed after transcription, before export out of the  mRNA is translated without any nucleus additional processing The link between nucleic acids and proteins  Is the GENETIC CODE  Cells use the genetic code to translate DNA/RNA sequences into proteins  The code was cracked by scientists in the 1960s Using logic to work out the GENETIC CODE  In DNA there are 4 nucleotides A, T, G, C 4 “letters” available to write the code  In RNA there are 4 nucleotides A, U, G, C  IN PROTEINS there are 20 different AAs  How might 4 LETTERS be arranged to spell 20 different WORDS?  Nucleotide code words of 1 letter - could only translate into 4 AAs (one letter – one AA)  Nucleotide words of 2 letters - could translate into 4x4 = 16 AAs (4 possibilities in the 1st position, each having 4 possibilities in 2nd position) Nucleotide words of 3 letters - … ?  If nucleotides are grouped in 3s , they can be used to spell 64 different words (4 3 )  More than enough to specify each of the 20 amino acids in proteins  SCIENTISTS CONCLUDED THAT  The DNA language (written using bases A, G, C, and T) must be written in words three nucleotides long  TRANSCRIBED into CODONS (again 3 bases long) on the mRNA (written using bases A, G, C, and U)  CODONS can then be TRANSLATED into the 20 amino acids that make up proteins By mid-1960s the code had been cracked  All 64 mRNA codons had been deciphered  Codons for all the 20 different amino acids were identified  You do not need to memorize the Table – it will Fig. 17.6 The Codon Table be given, but have to know how to read it) for mRNA We have 64 triplets and only 20 AA to code: 1.No ambiguity (no two AA are described by the same triplet) 2. Redundancy (all except Met and Trp described by more than one (64 combination for only 20 Aas…) 3. Punctuation (one Start and 3 Stop signs) Punctuation is important because it ensures the correct reading frame  Imagine a message sequence: thebigdogatetheredhat  Correct reading frame (i.e.: start in the right place, stop in the right place): the big dog ate the red hat  Incorrect reading frame – one off from the beginning: heb igd oga tet her edh at ◼ Conclusion - Start and Stop codons are vital  Universality of the Genetic Code  The genetic code is pretty much universal  It is shared by all organisms  from the prokaryotes, to the most complex plants and animals ◼ With only very minor exceptions Universality of the Genetic Code has: 1. Evolutionary significance:  A language shared by all living things must have been established very early in the history of life  Early enough to be present in the common ancestor of all living organisms  2. Practical significance: BIOTECHNOLOGY  If the same code can be transcribed and translated to give the same protein product in all organisms => Then genes can be moved between organisms, and the same protein products should still be expressed  GENE TRANSFER TECHNOLOGY has many applications  E.g. use of bacteria to express proteins with medicinal use. E.g.: Golden rice ◼ Insulin ◼ Human growth hormone Fig. 20.4 Quote from 1958 Nobel Laureate George Beadle and wife Muriel Beadle “Deciphering the DNA code has revealed a language much older than hieroglyphics, a language as old as life itself, a language that is the most living language of all- even if its letters are invisible and its words are buried deep in the cells of our bodies.”(1966)  Fig. 17.5 – The triplet code. The flow of information DNA RNA PROTEIN  1. DNA template strand acts as a transcription template  2. mRNA is transcribed as the exact complement of the DNA template sequence, except that URACIL substitutes for THYMINE.  3. Each group of three bases on the mRNA = a codon  4. Each codon is specific for a particular amino acid Concept 17.2 - Transcription is the DNA-directed synthesis of RNA Some transcription facts  Transcription = production of RNA (the “on-site instructions”) , from DNA (the “blueprint”)  RNA acts as an expendable, short-lived copy of the DNA blueprint  The nucleotides in DNA are A, T, G, and C  The nucleotides in RNA are A, U, G and C  They pair up like this Some transcription facts – cont.:  Gene - Definition = a region of DNA producing a functional product – either a polypeptide, or an RNA molecule (e.g. rRNA , tRNA)  Genes are found on both strands of the double helix. Strand A Strand B  Some transcription facts – cont.  For each gene, only one of the two DNA strands istranscribed  The strand transcribed = TEMPLATE STRAND  The one that isn’t transcribed = the non-template strand  Some transcription facts: directionality RNA is synthesized in an antiparallel direction to the DNA strand:  DNA is transcribed from 3’ to 5’end  while RNA is built 5’to 3’ - nucleotides are always added at the 3’ end of the mRNA as it grows Molecules involved in Transcription  DNA and RNA  RNA polymerase - the main enzyme responsible in RNA synthesis  FUNCTIONS of RNA polymerase –  opens up the two DNA strands  Works along the template strand in the 3’ to 5’ direction  Joins up RNA nucleotides (A, U, G and C) complementary to the sequence of DNA nucleotides on the template strand Fig. 17.8 - The stages of transcription  1. Initiation.  With establishment of the transcription initiation complex complete, transcription can start.  RNA Polymerase starts to unwind the DNA Fig. 17.8 - The stages of transcription  2. Elongation  RNA polymerase moves downstream along the DNA, unwinding it and synthesizing RNA.  Double helix is re-formed as RNA polymerase moves on ◼ Note directionality If a protein is needed in large amounts  Several RNA polymerase molecules can be transcribing the same gene at the same time! Fig. 17.8 - The stages of transcription. 3. Termination  When the end of the gene is reached, RNA polymerase must stop transcribing and detach.  Method of detaching is different in Prokaryotes and Eukaryotes  mRNA production - Summary  Three stages - Initiation Elongation Termination  Transcription starts at the start point, within the PROMOTER region of the gene  RNA polymerase adds nucleotides to the growing mRNA at the 3’ end  till a TERMINATION signal is reached and transcription is stopped  These stages of mRNA production apply to PROKARYOTES and EUKARYOTES  but after the RNA transcript is produced there are differences What happens AFTER the termination of the transcription Prokaryote mRNA needs no additional processing  It is ready to translate straight away  And because Prokaryotes have no nuclearmembrane ◼ mRNA is in contact with translation machinery of the cell as soon as it is transcribed  Result - translation can begin before transcription is complete PROKARYOTES Advantage: simultaneous transcription and translation can produce lots of protein from a single gene Concept 17.3 – Eukaryotic Cells Modify RNA After Transcription Unlike the prokaryotes Eukaryotes produce pre-mRNA which is processed to mRNA - done still inside nucleus (i.e. within the nuclear membrane) - Ony then exported into cytoplasm  What does the processing involve? 1. Both ends of the mRNA are modified  5’ end is capped with modified guanine nucleotide (= “ 5’ cap“ )  3’ end has lots of adenines added to the AAUAAA already present (= “poly-A tail“) Cap Tail 5’ untranslated region 3’ untranslated region What the 5’ cap and the poly-A tail: are good for?  A) Help with exporting of mRNA from nucleus to cytoplasm (may be recognized by the transporters?)  B) Protect mRNA from early attack by hydrolytic enzymes – how?  C) Help ribosomes in cytoplasm attach to the right end of the mRNA (5’ end) for correct translation (see the next subsection) What else can we see in that Figure 17.11? Only a small part of the mRNA includes the protein-coding segments Cap Tail 5’ untranslated region 3’ untranslated region And even in this region, big chunks between the start and stop codons also don’t code for the polypeptide 2. Eukaryotic genes have long non-coding stretches called INTRONS that do not code for proteins and MUST BE REMOVED inside the nucleus, before translation outside the nucleus RNA transcript coding for -globin, which is 146 amino acids long, therefore mRNA needs 146 codons (438 bases)  Introns are intervening sequences that must be cut out. They will stay in the nucleus and be broken down to nucleotide monomers  Exons are the bits you want  Exons are the regions that will be expressed and contribute to the final polypeptide. They will exit the nucleus  Introns are removed by splicing  Splicing is achieved by spliceosomes ◼ Large complexes made of protein and small RNA molecules  The small RNA molecules ◼ participate in spliceosome assembly ◼ Bind onto the mRNA and Fig. 17.13 identify the introns to be cut out ◼ and catalyse the splicing reaction  In other words – they act as enzymes!  Why do we have introns?  Some of them may be a by-product of “alternative RNA splicing” Depending on what is cut out (i.e. what you consider intron) one gene can encode more than one polypeptide  If I have a word “Catastrophe” I could use its portion to describe also “at”, “Cat”, “strophe” (the first section of an ancient Greek choral ode) etc. ◼ Results from the Human Genome Project suggest that alternative RNA splicing is one reason why humans are more complex than roundworms (nematodes) despite having practically the same number of genes as a nematode!! (i.e. we use some genes to produce several different products)  Why do we have introns?  Other introns may be not our DNA , but “parasitic DNA”/”selfish DNA”/”Genomic outlaws” – pieces of DNA that at some point in the past brought by viruses or picked from environment –inserted themselves into DNA of our ancestors and get replicated during each cell division ever since.  So why we have not got rid of them?  Either our cells don’t have mechanisms to get rid of them  Or there may be redeeming benefits:  Possible usefulness during meiosis:  Introns provide spaces between the coding regions making crossing over easier and less likely to disrupt a functional gene sequence – if the break happens in an intron – no damage to the exon genes Fig. 17.4 Overview of the roles of transcription and translation inthe flow of genetic information Transcription is now complete And we are here  Concept 17.4 - Translation is the RNA-directed synthesis of a polypeptide: A closer look  Translation = changing from one language to another ◼ from  Language of Nucleotides ◼ ACGAAAGGU  into  Language of Amino Acids: ◼ Thr –Lys -Gly Building a polypeptide  Translation – the players 1. mRNA ◼ Which exits the nucleus after secondary processing mRNA 2. Transfer RNA (tRNA) – a molecule with two special sites  Amino Acid attachment site  Anticodon that matches a specific mRNA codon Fig. 17.16 - The structure of transfer RNA (tRNA) 3. Enzymes called Aminoacyl-tRNA synthetases: attach the right amino acids to their tRNA molecules Figure 17.17  There are 20 different amino acids  and 20 aminoacyl-tRNA synthetase enzymes  each aminoacyl-tRNA synthetase enzyme has an ACTIVE SITE  specific for one amino acid and  and all the different tRNAs with anticodons for that particular amino acid Using ATP, the enzyme (generic name = aminoacyl-tRNA- synthetase) links tTNA and its amino acid together Charged tRNAs are now ready for the process of translation  Ribosomes are the next requirement ◼ Protein synthesis is very important so … ◼ Most cells contain 1000s of Ribosomes  Prokaryotes and Eukaryotes both have Ribosomes  Composed of ~2/3 RIBOSOMAL RNA (rRNA) and 1/3 PROTEINS ◼ Proteins mainly on the outside  Come as two sub-units (large and small) that lock together over the mRNA  Bacterial and Eukaryotic ribosomes are quite similar  But they do have some minor differences:  Eukaryotic ribosomes are a bit larger  There are some minor differences in molecular composition  This is very useful: some antibiotics e.g. tetracycline, streptomycin target bacterial ribosomes without harming human ones … Recap - MAKING A RIBOSOME in a Eukaryotic cell In Eukaryotes,  rRNA is transcribed in the NUCLEOLUS  Production of ribosomal proteins takes place in the cytoplasm (outside the nucleus)  Ribosomal proteins are then IMPORTED into the nucleus  RIBOSOMAL SUBUNITS are ASSEMBLED in the NUCLEOLUS  And exported to the cytoplasm, where they do their work  RIBOSOME STRUCTURE  All ribosomes have a large and a small subunit  Made of rRNA (2/3) and protein (1/3)  With proteins on the outside  Ribosomes have 3 sites - A, P and E  and an exit tunnel (for the polypeptide to grow through) Fig. 17.18 Anatomy of a functioning ribosome Most of the work done by the ribosome is actually done by the rRNA  Ribosomal RNA is responsible for 1) positioning the mRNA and tRNAs correctly 2) positioning the amino acids so that they can be added to the polypeptide chain as it grows 3) catalyzing peptide bond formation  A ribosome can be thought of as one huge RIBOZYME! Building a Polypeptide: Step 1 - Initiation Small ribosomal subunit Initiator tRNA with All come methionine attached together The mRNA And:  mRNA attaches to the small subunit,  the initiator tRNA binds to the START codon on the mRNA,  ensuring the correct READING FRAME  Large ribosomal subunit attaches over the mRNA  Putting the tRNA in the P site The process is slightly different between bacteria and eukaryotes, but the end result is the same: READY FOR TRANSLATION !  Building a Polypeptide: Step 2 - Elongation  tRNAs pass sequentially through the ribosome’s 3 binding sites  In the A site (aminoacyl-tRNA site - Attachment site), the anticodon on the incoming tRNA binds to the codonon mRNA in the vacant A site  While in the A site, rRNA in the large subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the growing polypeptide chain sitting in the P site ◼ The P site (peptidyl-tRNA site - Polypeptide building site), The growing polypeptide is transferred to the tRNA in the A site  Everything shifts along one place  The tRNAs in the P and A sites, plus the mRNA they are attached to are all shifted along one place in the ribosome  TRANSLOCATION  The empty tRNA is now in the E site – EXIT site  tRNA leaves the ribosome to pick up another AMINO ACID Figure 17.20 – The Elongation Cycle of Translation A new tRNAarrives 1 in the Asite This goes on until the polypeptide 2 is complete 4 3 Step 3 - Termination  Eventually, a STOP codon (UAA, UAG, UGA) on mRNA arrives in the A site  A release factor protein (not another tRNA) binds to the STOP codon, and  Cuts the polypeptide free of the last tRNA  The completed polypeptide is released ◼ and departs through the exit tunnel of the ribosome’s large sub-unit End Result - Production of Polypeptides (Proteins)  The job is done; the ribosome subunits come apart Fig. 17.21 - The termination of translation  Figure 17.25 - A summary of transcription and translation in a eukaryotic cell Self Study  Completing and Targeting the Functional Protein (pages 376-377)  Making multiple Polypeptides in Bacteria and Eukaryotes (pages 377-378) Check out the animation “Protein Synthesis” in the Animations folder on our Brightspace  Plus this good you tube video of transcription and translation  DNA transcription and translation [HD animation]  https://www.youtube.com/watch?v=8_f-8ISZ164