Section 7. Junaid Malek, M.D.

Transcription

1 Section 7 Junaid Malek, M.D.

2 RNA Processing and Nomenclature For the purposes of this class, please do not refer to anything as mrna that has not been completely processed (spliced, capped, tailed) RNAs that will become mrnas should either be called pre-mrnas, primary transcripts, or RNAs

3 5 Capping What you need to know: Essential for nuclear transport Probably enhances mrna stability

4 5 Capping Don t need to know mechanism Note that two of the three phosphate groups in the RNA cap come from the RNA chain, while only one comes from the capping nucleotide H 2 N N HN O OH OH H H H H O O O O O P O P O P O - N O - O - O - N CH 3 - O NH 2 N N O O O N N P O P O P O O NH 2 O - O - O - H H H H N OH O N O O P O O O - H H O H H OH NH O N O O P O O O - H H H H OH H 2 N N HN O OH OH H H H H O O N N CH 3 O P O - O O O N O P O P O P O O O - O - O - H H H H OH O O P O O - N NH 2 N N NH 2 N O H H H H OH O O P O O - N O O N O H H H H OH NH O O O P O O - O O P O O -

5 3 Poly-adenylation Specifics won t be tested Know that poly-adenylation is performed by a protein that recognizes a specific sequence on the RNA transcript When it binds, it then starts adding A s onto the 3 end A common polya signal: 5 -AAUAAA-3

6 Q: You isolate a mrna from a eukaryotic cell and hybridize it to genomic DNA and see the following: Red=RNA Blue=DNA Explain the regions of hybridization

7 RNA Features Splicing 5 exon/intron splice junction 3 exon/intron splice junction branchpoint sequence Other necessary molecules small nuclear Ribonucleoprotein Particles (snrnps) Branchpoint Binding Protein (BBP)

8 Splicing Branchpoint 2 -OH on adenosine attacks 5 exon to form lariat 3 -OH of the 5 exon attacks the phosphate of the guanosine at the 3 exon to fuse the two and liberate the lariat

9 Q: There are an estimated 30,000 genes in the human genome, but over 100,000 different transcripts have been identified. How can this be? A: Alternative splicing of a gene with multiple exons can generate many different transcripts

10 REV, RRE and Nuclear Export Q: How can HIV make so many different gene products with a compact (9kb) genome? A: By using REV to export different forms of the HIV transcript into the cytoplasm to be transcribed

11 Rev and RRE The HIV mrnas are produced from a primary transcript by three different splicings: unspliced, singly spliced and doubly splice Although unspliced and singly sliced mrnas are made before doubly spliced mrnas, the protein products of the doubly spliced mrnas are the first synthesized in the cytoplasm because they are smaller and exported faster Expression of unspliced and singly sliced mrnas would be negligible without the Rev protein, which is a product of doubly spliced mrna

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13 Rev and RRE REV is an RNA binding protein that binds to a specific RNA sequence referred to as the REV response element (RRE) RRE is normally in an intron and should be spliced out prior to nuclear export of the RNA REV can bind to the RRE on other HIV transcripts that have not yet been spliced or completely spliced and then recruit other proteins (Xpo and Ran) to export the unspliced or incompletely spliced transcript out of the nucleus

14 Why is any of this important? The presences of these two other transcripts in the cytoplasm allows for the translation of different genes This also allows for the expression of different genes at different times as it takes time for REV to be transcribed and translated

15 It s all about timing... Early: 2 kb transcript (completely spliced) encodes Rev and Tat key for enhancing transcription (Tat) and inducing expression of later genes (Rev) Later: 4 kb and 9 kb transcripts (incompletely spliced and unspliced) encodes later genes needed for the manufacture of additional viral particles: gag, env, pol (protease/integrase/rt)

16 Translation RNA-directed synthesis of proteins Three classes of RNA are required to synthesize proteins mrna= informational template trna= molecular adaptors that match AA to mrna code rrna=help form ribosomes with proteins

17 The Genetic Code 4 different nucleotides encode for 20 amino acids nucleotide triplet in mrna=codon codons read on mrna from 5 to 3 reading frame established by start position of first codon mrna code can be translated in one of three reading frames Proteins are translated in a specific reading frame

18 NOTE: You are not responsible for memorizing the genetic code for the exam (it will be provided for you) However, it is useful to be able to recognize the start codon (AUG for Met) and the stop codons (UAA, UAG, UGA)

19 trna 2 key domains anticodon= nucleotide triplet that basepairs with mrna codon 3 end= attachment site for aa

20 Q: Can trnas recognize more than one codon? A: Yes, mismatch at 3 position occurs Q: Can aa have more than one trna? A: Yes

21 Aminoacyl-tRNA Synthetase protein enzyme that couples trna with correct AA Each aminoacyl-trna synthetase recognizes one AA and all of its matching trnas

22 Aminoacyl-tRNA Synthetase 2 pockets on synthetase help ensure correct coupling Synthesis site excludes amino acids that are too large Editing site excludes correct amino acid, but accepts and removes incorrect amino acids that are similar in size

23 The Ribosome Large complex of protein (1/3) and RNA (2/3) In eukaryotic cells, ribosomal subunits are assembled at the nucleolus, by the association of newly transcribed and modified rrnas with ribosomal proteins, which have been transported into the nucleus after their synthesis in the cytoplasm. The two ribosomal subunits are then exported to the cytoplasm, where they perform protein synthesis. 2 subunits called large and small. The two subunits come together on an mrna usually near its 3 end to begin synthesis of protein.

24 The Ribosome 3 sites in ribosome bind to trna A-site: binds aminoacyl-trna (A=acceptor) P-site: binds peptidyltrna E-site: binds exiting trna (E=exit)

25 Protein Translation Cycle Step 1: Incoming amino acid + trna is selected based on anticodon to codon base pairing at the A-site Step 2: The bond between the end of the amino acid chain and the trna at the P-site is broken. The free end of the amino acid chain is then bonded to the amino acid of the trna in the A-site. Ribosome shifts down the mrna by three nucleotides, placing the trnas in the E- and P-sites Step 3: The spent trna is ejected and the ribosome is reset to bind another amino acid + trna at the A-site

26 Q: In an experiment performed in 1962, a cysteine already attached to its trna was chemically modified to an alanine. If you used this hybrid trna in a cell free translation system where the normal cysteine-trnas were removed, what would you predict would happen? A: At the mrna codons for cysteine, alanine would be added to the polypeptide chain instead of cysteine NOTE: The ribosome itself has no proofreading function. Translational accuracy relies on the aminoacyl-trna synthetase attaching the correct amino acid to the corresponding trnas and the binding between the trna anticodon and the mrna codon. This accuracy of this step is aided by elongation factors.

27 EF-Tu The aminoacyl-trna is bound to EF-Tu-GTP when it enters the A site of the ribosome As long as EF-Tu-GTP is present a peptide bond cannot form between the amino acid on the incoming trna and the growing polypeptide chain GTP must first hydrolyze to GDP for peptide bond formation to be allowed (1st delay)

28 EF-Tu trnas with an codon that correctly base pairs with the mrna codon will remain bound long enough for hydrolysis of GTP to GDP to occur and for the amino acid to add onto the growing polypeptide chain trnas with an anticodon that does not correctly base pair with the mrna codon will have time to dissociate The rate of GTP hydrolysis by EF-Tu is actually faster for a correct codon-anticodon pair than for an incorrect pair; this provides an even longer window of opportunity for incorrectly bound trna molecule to dissociate from the ribosome

29 EF-Tu Once EF-Tu-GTP is hydrolyzed into EF-Tu- GDP, it dissociates from the aminoacyl-trna allowing the trna to be fully accommodated into the A-site (2nd delay)

30 EF-G EF-G-GTP binds near the A-site EF-G accelerates the movement of the two bound trnas into the A/P and P/E hybrid states Contact with the ribosome stimulates the GTPase activity of EF-G, causing a dramatic conformational change in EF-G as it switches from the GTP to the GDP-bound form

31 EF-G This change moves the trna bound to the A/P hybrid state to the P-site and advances the cycle of translation forward by one codon

32 Elongation Factors During each cycle of translation elongation, the trnas molecules move through the ribosome in an elaborate series of gyrations during which they transiently occupy several hybrid binding states In one, the trna is simultaneously bound to the A site of the small subunit and the P site of the large subunit; in another, the trna is bound to the P site of the small subunit and the E site of the large subunit

33 Elongation Factors In a single cycle, a trna molecule is considered to occupy six different sites, the initial binding site (called the A/T hybrid state), the A/A site, the A/P hybrid state, the P/P site, the P/E hybrid state, and the E-site. Each trna is thought to ratchet through these positions, undergoing rotations along its long axis at each change in location

34 Elongation Factors EF-Tu and EF-G are the designations used for the bacterial elongation factors In eukaryotes, they are called EF-1 and EF-2, respectively Changes in the three-dimensional structure of EF-Tu are caused by GTP hydrolysis For each peptide bond formed, a molecule of EF-Tu and EF-G are each released in their inactive, GDP-bound forms

35 Elongation Factors To be used again, these proteins must have their GDP exchanged for GTP In the case of EF-Tu, this exchange is performed by a specific member of a large class of proteins known as GTP exchange factors

36 Alberts MBOC Figure 6-66

37 Initiation of Translation 1) Special initiator-trna (coupled to AA Met) binds to small ribosomal subunit 2) Small ribosomal subunit binds 5 end of mrna molecule. mrna is recognized by its 5 cap and bound initation factors (IF). 3) Small ribosomal subunit moves 5 to 3 on mrna until it finds an AUG codon. This movement is facilitated by additional IFs that use ATP. 4) Once it finds the AUG some IFs dissociate and the large ribosomal subunit binds such that the initiator trna is bound to the P-site and the A-site is vacant. 1) The translation cycle now begins

38 What about Initiation in Bacteria? The mechanism for selecting a start codon in bacteria is different. Bacterial mrnas have no 5 caps to tell the ribosome where to begin searching for the start of translation. Instead, each bacterial mrna contains a specific ribosome-binding site (called the Shine-Dalgarno sequence, named after its discoverers) that is located a few nucleotides upstream of the AUG at which translation is to begin. This nucleotide sequence, with the consensus 5 -AGGAGGU-3, forms base pairs with the 16S rrna of the small ribosomal subunit to position the initiating AUG codon in the ribosome. A set of translation initiation factors orchestrates this interaction, as well as the subsequent assembly of the large ribosomal subunit to complete the ribosome. Unlike a eucaryotic ribosome, a bacterial ribosome can therefore readily assemble directly on a start codon that lies in the interior of an mrna molecule, so long as a ribosome-binding site precedes it by several nucleotides. As a result, bacterial mrnas are often polycistronic that is, they encode several different proteins, each of which is translated from the same mrna molecule. In contrast, a eucaryotic mrna generally encodes only a single protein. -Alberts MBOC

39 Translation Termination Translation termination uses release factors, which are an example of molecular mimicry The three-dimensional structure of release factors (made entirely of protein) bears an uncanny resemblance to the shape and charge distribution of a trna molecule This shape and charge mimicry allows the release factor to enter the A-site on the ribosome and cause translation termination

40 Translation Termination: Overview 1) Stop codon (UAA, UAG, UGA) in mrna is a signal for translation termination 2) The stop codon in the A-site is bound by a release factor 3) Binding of release factor forces the peptidyl transferase in the ribosome to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-trna. This releases the amino acid chain from the ribosome. 4) The ribosome complex dissassembles