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Chapter 12 RNA and Protein Synthesis: The Expression of Genetic Information Lecture Outline I. DNA is transcribed to form RNA; RNA is translated to form proteins A. RNA (ribonucleic acid) is used as an intermediary between DNA and proteins 1. RNA is a single-stranded nucleotide polymer (although some areas may be folded) 2. The sugar in RNA is ribose, and uracil substitutes for thymine Uracil is a pyrimidine and forms two bonds with adenine Difference between DNA and RNA structures (See figures be Characters DNA R 1. Molecule Double stranded, helical 2. Pentose sugar Deoxyribose Rib 3. Pyrimidine base Thymine Ur 4. Complementary base pairing Always present and exists between A = T and G = C Single stranded, straight or Normally absent, but m segments o If present, pairing is be 5. Ratio of Purines: Pyrimidines Always 1:1 B. A copy of the DNA is made in the form of messenger RNA (mRNA) in transcription Not nece C. Translation involves mRNA, tRNA (transfer RNA), and rRNA (ribosomal RNA) coordinating to produce proteins D. mRNA has sequences of 3 nucleotides called codons E. Codons are read in sequences of 3; this is called triplet code Codons are written in 5’ to 3’ fashion F. Each codon codes for 1 amino acid Four bases can combine in 43 combinations (64) – more than enough to code for the 20 naturally occurring amino acids G. Each tRNA molecule has a sequence of 3 nucleotides – the anticodon 1. Anticodons base pair with a codon in a complimentary way 2. Anticodons are written in a 3’ to 5’ fashion H. Ribosomes are composed of proteins and rRNA II. Transcription is the synthesis of RNA from a DNA template A. RNA is synthesized by DNA – dependent RNA polymerases, which are similar to DNA polymerases B. Messenger RNA contains base sequences that code for a protein 1. Transcription does not involve a primer; it begins at a promoter sequence on the template strand (the promoter is not transcribed) 2. RNA synthesis proceeds in a 5’→3’ direction (copying the DNA from the 3’→5’) a. Upstream refers to sections towards the 5’ end of the mRNA sequence b. Downstream refers to sections towards the 3’ end of the mRNA sequence c. Bacterial promoters are about 40 bases long and are located in the DNA just upstream from the starting point d. Sequences at the end of the gene act as ‘stop’ signals 3. Typically only one strand of the DNA is transcribed – the template strand In a single DNA molecule, some of the template sections are on one strand, others are on other strands For an animation of mRNA transcription go to: http://www.google.com/imgres?imgurl=http://www.virtualsciencefair.org/2004/m cgo4s0/public_html/t3/RNAstructure1.jpg&imgrefurl=http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t 3/RNA.html&h=351&w=292&sz=12&tbnid=30TvlbQZWGYJ::&tbnh=120&tbnw=100 &prev=/images%3Fq%3Drna%2Bstructure%2Bimage&usg=__HWdESNwYOlwzGpiK x7LB3aWklWA=&sa=X&oi=image_result&resnum=3&ct=image&cd=1 C. Messenger RNA contains additional base sequences that do not directly code for protein 1. mRNA has a noncoding leader sequence at the 5’ end, important in binding to the ribosome during translation 2. Coding sequences follow the leader sequence 3. Coding sequences are followed by termination signals and other noncoding sequences III. During translation, the nucleic acid message is decoded A. An amino acid is attached to transfer RNA before becoming incorporated into a polypeptide 1. To form a polypeptide chain, the amino and carboxyl groups of amino acids are joined 2. The specific sequence of the amino acids (primary structure) is dictated by the sequence of codons of the mRNA 3. tRNA molecules are produced from tRNA genes a. tRNA is linked to amino acids by aminoacyl-tRNA synthetases b. This is an energy-requiring process B. Transfer RNA molecules have specialized regions with specific functions 1. tRNA molecules have attachment sites for amino acids 2. tRNA molecules have anticodons which will bind to the mRNA 3. tRNA molecules must be recognized by both the specific aminoacyl-tRNA synthetase and the ribosome 4. tRNA molecules are approximately 70 nucleotides long, with some generic sections and some unique sections 5. The nucleotide chain is folded back upon itself to form 3 or more loops with unpaired nucleotides exposed C. The components of the translational machinery come together at the ribosomes Ribosomes are composed of two subunits 1. In E. coli, the small subunit is composed of 21 proteins and one RNA molecule, the larger subunit is composed of 35 proteins and two RNA molecules 2. The large subunit has a groove into which the small subunit fits 3. Ribosomes are transcribed from DNA, but do not carry information – rather they function as the physical site of translation, and as a catalyst 4. The A site of the ribosome is where the aminoacyl-tRNA binds 5. The P site of the ribosome is where the tRNA holding the polypeptide chain is positioned D. Translation includes initiation, elongation, and termination To view an animation of translation, including initiation, elongation, and termination click the link below. Protein Synthesis: at the ribosome http://www.sciencenetlinks.com/interactives/protein.html 1. Initiation is the first step a. Initiation factors (proteins) move initiation tRNA onto the small ribosomal subunit b. The codon for the initiation is AUG, which codes for the amino acid methionine (met) c. The initiation complex binds to ribosomerecognition sequences on the mRNA, and aligns the anticodon of the tRNA with the codon of the mRNA d. The large ribosomal subunit then binds, forming the functional ribosome 2. Elongation is the addition of new amino acids a. As the initiator tRNA is bound to the P site of the ribosome, the A site is unoccupied until the next aminoacyl-tRNA moves in b. The energy for this process comes from guanosine triphosphate (GTP) c. Peptide bond formation takes place between the amino group of new amino acid and the carboxyl group of the amino acid bound in the preceding reaction d. Protein synthesis proceeds from the amino acid end to the carboxyl end e. The tRNA molecule is released from the P site This reaction requires the ribosome, peptidyl transferase f. Translocation is the movement of the growing polypeptide chain from the A site to the P site Energy for translocation comes from GTP g. Translation of the mRNA proceeds from 3’ to a 5’ direction, which is the same direction as transcription h. Termination occurs when the mRNA presents the codons UAA, UGA, or UAG, as there is no tRNA which is complimentary to them 1). Release factors recognize these codons 2). The ribosome dissociates into the two subunits At right are images of the E. coli ribosome, reconstructed from electron density maps obtained by threedimensional cryo-electron microscopy. TWO VIEWS OF THE STRUCTURE OF THE THERMUS THERMOPHILUS 70S RIBOSOME AT 7.8 � RESOLUTION. TRANSFER RNAs (YELLOW, GREEN, and RED) OCCUPY A CAVITY BETWEEN TWO RIBOSOMAL SUBUNITS. E. A polyribosome is a complex of one mRNA and many ribosomes 1. In prokaryotes, transcription and translation simultaneously occur 2. The half-life of the mRNA molecules in Bacteria is about 2 minutes IV. Transcription and translation are more complex in eukaryotes than in prokaryotes A. Eukaryotic mRNA molecules are processed after transcription 1. Processing occurs in the nucleus of eukaryotes 2. As the mRNA transcript grows, a cap is added to the 5’ end a. The cap is 7-methylguanylate b. The cap is necessary for binding to ribosomes 3. Capping may protect mRNA from degradation; half-lives of eukaryotic mRNA molecules are as long as 24 hours 4. At the 3’ end of the molecule, a sequence is added which acts as a signal for the subsequent addition of a “tail” 5. Enzymes recognize the signal and add 100-250 adenines (the poly-A tail) B. Both noncoding sequences and coding sequences are transcribed from eukaryotic genes 1. Coding sequences are exons (expressed sequences); noncoding sequences are introns (intervening sequences) This terminology refers to sequences in both DNA and mRNA 2. Precursor mRNA (pre-mRNA) contains both introns and exons 3. Small nuclear ribonucleoprotein complexes (snRNPs) may bind to the introns and catalyze excision and splicing 4. RNA may also act as self-acting enzyme and excise and splice without the intervention of protein enzymes V. The genetic code is read as a series of codons in mRNA A. In 1961 Crick and colleagues determined that the code was read in a non-overlapping sequence of bases, forming a 3 base reading frame 1. Niremberg and Mathaei synthesized poly-U (UUUUUUU…) and found that it coded for phenylalanine 2. UAA, UGA, UAG were found to be stop codons, which do not code for any amino acids 3. AUG is the start codon and codes for Methionine 4. The genetic code of all 64 possible codons has been found to be nearly universal B. The genetic code is redundant 1. Redundant codons have similar base sequences 2. Only methionine (AUG – start codon) and tryptophan (UGG) have single triplet codes 3. The wobble hypothesis, proposed by Crick, suggests that the third nucleotide Of the tRNA anticodon can bind with more than one nucleotide of the mRNA codon, as cells typically produce only about 40 different tRNA molecules VI. A gene is defined as a functional unit A gene is a transcribed nucleotide sequence that yields a product VII. Mutations are changes in DNA A. Base-substitution mutations involve the substitution of one base for another 1. Due to redundancy of the code, this may not alter the product 2. Missense mutations result in the substitution of one amino acid for another The resulting protein may be nonfunctional, or the mutation may have no effect on the organism (a silent mutation) 3. Nonsense mutations result in a termination codon B. Frameshift mutations involve insertion or deletion of a base Frameshift mutations result in an entirely different sequence of amino acids (they change the reading frame) C. Transposons are movable sequences of DNA which may move into another area of DNA 1. Transposons may disrupt genes, but may also activate some genes 2. Transposons were discovered by Barbara McClintock in corn in the 1950s (she received the Nobel Prize in 1983) 3. Have some similarities to retroviruses D. Hot spots are regions of DNA more likely to undergo mutations, and are often regions of repeated nucleotides, causing the polymerases to “slip” E. Mutagens are agents which cause mutations, including ionizing radiation 1. Mutations in somatic cells are not passed on to the next generation 2. Some mutagens are also carcinogens