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Transcription continued: slide 2 . Catalyzed by RNA polymerase, which can do the following:1. Separate the DNA strands and link RNA nucleotides along the DNA template2. Add Nucleotides only to the 3' end3. Prokaryotes have one type of polymerase whereas eukaryotes (euk's) have three typesa. i.e.

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    1. Transcription Purpose: RNA is transcribed from DNA to a complementary nucleotide sequence in messenger RNA (mRNA)

    2. Transcription continued: slide 2 Catalyzed by RNA polymerase, which can do the following: 1. Separate the DNA strands and link RNA nucleotides along the DNA template 2. Add Nucleotides only to the 3 end 3. Prokaryotes have one type of polymerase whereas eukaryotes (euks) have three types a. i.e.., RNA polymerase II catalyzes mRNA synthesis; It transcribes genes that will be translated into proteins

    3. Transcription continued: Slide 3 Has a transcription unit that includes a nucleotide (nucle) sequence on the template strand of DNA; includes initiation and termination sequences as well as the nucleotides between. 1. In eukaryotes it includes one gene 2. In prokaryotes it can contain several genes

    4. Transcription: slide 4 Binding and Initiation of mRNA includes: 1. RNA polymerase binding to regions called promoters 2. Includes the initiation site (~100 nucleotides long) 3. In euks, polymerases cannot bind w/t help of transcription factors a. i.e., TATA box = nucleotides sequence at the promoter that is rich in T and A; ~25 nucles upstream from the initiation site

    5. Transcription: Slide 5 4. When the binding occurs, the enzyme separates the 2 DNA strands and transcription begins Elongation of the RNA strand occurs 1. As the RNA polymerase II moves along the DNA it does 2 functions a. untwists and opens a short segment of DNA of ~10 nucles b. links the new nucleotide one at a time in the 5 to 3 direction

    6. Transcription: Slide 6 c. The growing RNA strands hang free from each polymerase d. The same gene maybe transcribed many times Termination of Transcription 1. Polymerase continues until a termination site occurs a. i.e., AATAA

    7. Transcription: Slide 7 2. Other enzymes are involved Euks modify RNA after transcription 1. Both ends are covalently altered a. 5 cap where guanine is added to the 5 end protects it from degradation by hydrolytic enzymes helps ribosomal subunits recognize the attachments site b. a poly A-trail is added (200 As) added to the 3 end

    8. Transcription: slide 8 C. Sometimes a leader sequence is attached, plus a trailer sequence - both noncoding sequences 3. The original RNA is heterogenous nuclear RNA a. After being processed, only a small portion of the original pre-mRNA will leave the mRNA

    9. Transcription: slide 9 b. only a small portion of this RNA will leave the nucleus as mRNA 4. Introns are those noncoding sequences in DNA that intervene between coding sequences (exons) which are transcribed but later excised thru a process of RNA splicing before leaving the nucleus

    10. Transcription: slide 10 Small nuclear ribonucleoproteins or snRNPs (snurps) recognize splice sites. Made of RNA & protein Called Spliceosomes Splice out the introns & join the exons together 6. Exons are the coding sequences that are transcribed and eventually leave the nucleus to be expressed through translation 7. Sometimes ribozymes (RNA molecules) function as enzymes Act as self-splicing enzymes Thus not all enzymes are proteins Notes that there is alternative RNA splicing in some species (fruit flies) & exon shuffling or increase chance of crossing over between exons and exons and exons and introns. Plus there are domains in the exons regions that code for different parts of a protein or even possibly different proteins altogether.

    11. How did they decipher mRNA? 1. Marshall Nirenberg (in 1961) synthesized the first a. Added only poly U to a test that had all the components for protein synthesis b. mRNA was translated into a polypeptide that only contained a string of amino acids that were phenylalanine c. Thus UUU specifies phenylalanine only

    12. Translation Translation = The Linear sequence of bases in mRNA is translated into the linear sequence of amino acids in a polypeptide 1. The mRNA is read in triplets, AUG, which is a codon message that signals the start of translation

    13. Translation: slide 2 2. The grouping of nucleotides is important in the molecular language of cells. This ordering is called the reading frame. a. UGGUUUGGCCGUUUU is read in a series of nonoverlaping three letter words: UGG-UUU-GGC-CGU-UUU This message becomes Trp-Phe-Cly-Arg-Phe when translated by the anticodon. 3. The transfer RNA (tRNA) is the interpreter between two forms on information - base sequence in mRNA and amino acid sequence in polypeptide

    14. Translation: slide 3 4. How does the tRNA translate the mRNA? a. the structure of a tRNA as seen on p. 320 has a anticodon region which fits onto the mRNA b. the other end of the tRNA molecule attaches to a specific amino acid c. the codon (three letter triplet code, i.e., UUA) matches up by H-bonding with the complementary triplet code on the anticodon The tRNA's decode the message, codon by codon f. i.e., UUU translates into phenylalanine. HOW?

    15. Structure and Function of tRNA g. The tRNA transfers phenylalanine to the ribosome that has an anticodon of AAA h. Thus phenylalanine will be added to the growing polypeptide at the P site on the tRNA i. As tRNAs deposit a.a.s in the correct order (A site on the tRNA), ribosomal enzymes link them into a chain at the P site 6. Structure and Function of tRNA a. All types of RNA (i.e., tRNA) are transcribed from DNA in the nucleus b. So the tRNA must travel from the nucleus to cytoplasm

    16. Structure and Function of tRNA contd c. each tRNA can be used repeatedly d. Its form fits function e. it is single-stranded (~80 nucles) f. folding of forms several double strands because H-bonding with other bases clover leaf shape g. 3-D shape is L-shaped h. Or clover leaf shape i. At the end of the L-shape is the three base sequence called the anticodon

    17. Structure and Function of tRNA contd k. ~45 distinct types of tRNA; enough to translate the 64 codons (some tRNA's can recognize 2 or 3 codons specifying the same a.a.) l. the previous statement is because of the wobble effect - the third base (5 end) of the tRNA can hydrogen bond with more than one kind of base (in the 3 end) of the codon i.e., U can wobble to pair with A or G. Some have inosine (I), which can then bond with U, C, or A or recognize GGU, GGC or GGA - all which code for glycine An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA. HOW? The ATP becomes AMP in an endergonic process

    18. Building of a polypeptide This consists of three stages: 1) initiation; 2) elongation; 3) termination. 1. Initation a. small ribosomal subunit binds to the AUG of the codon with an anticodon UAC. What A.A. is laid down? Thus the AUG is a start codon where translation usually starts. b. The small ribosomal subunit moves down the mRNA and binds to the 5 end. It then bonds with the complementary sequence of rRNA

    19. Function of Ribosome The mRNA, initiator tRNA and the small ribosomal subunit are first connected, then the attachment of the large ribosomal subunit occurs using GTP for energy to form the initiation complex

    20. Ribosomal Structure cont. A site (aminoacyl-tRNA binding site) where the next A.A. is brought into position on the mRNA codon by the tRNA P site (peptidyl-tRNA binding site) is where the tRNA, carrying the growing polypeptide chain, accepts the new A.A. from the A site E site or exit site is where the previous tRNA that carried the polypeptide chain leaves the ribosome

    21. Elongation 2. Elongation (three steps) a. Codon recognition A site forms hydrogen bonds w/anticodon b. Peptide bond formation an enzyme, peptidyl transferase, catalyzes a peptide bond between the a.a.s of the P site and the new a.a. in the A site the new polypeptide separates from its tRNA and connects to the new a.a. at the A site.

    22. Elongation Contd C. Translocation the tRNA in the P site releases from the ribosome, and the tRNA in the A site is translocated to the P site the mRNA moves through the ribosome in the 5 to 3 direction GTP molecule provides energy for each translocation steps A protein call the release factor binds to the stop codon (i.e., UAA, UGA, or UAG) and water is replaces the AA with a water molecule

    23. Elongation cont. See fig. 17.18 on p. 324 Several ribosomes can be translating the message at one time called polyribosomes (or polysomes). Proteins or polypeptides chains detach and are sometimes modified in the cell before being used (one protein becomes two, i.e. insulin)

    24. SRP SRP is a signal-recognition particle which Has a signal built into it that bonds to a newly made polypeptide that is told to bring the protein and perhaps a ribosome to the ER to be be used or processed by the following: a. Inside the ERs lumen b. In the mitochondria c. In the chloroplast d. In the nucleus e. In other organelles f. Or secretion

    25. Multiple RNAs mRNA tRNA rRNA Primary transcript snRNA (small nuclear RNA) SRP RNA (signal-recognition particle) snoRNA (Small nucleolar RNA)

    26. Web sites for animation of transcription & Translation Replication/Transcription/Translation http://vcell.ndsu.nodak.edu/animations/transcription/TranscriptionAdvanced.wmv

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