From RNA To Protein

Transcription

1 From RNA To Protein

2 Introduction mrna Processing heterogeneous nuclear RNA (hnrna) RNA that comprises transcripts of nuclear genes made by RNA polymerase II; it has a wide size distribution and low stability. pre-mrna The nuclear transcript that is processed by modification and splicing to give an mrna. RNA splicing The process of excising introns from RNA and connecting the exons into a continuous mrna.

3 hnrnp= (heterogeneous nuclear RNA), in which the hnrna is complexed with proteins. Pre-mRNAs are not exported until processing is complete; thus they are found only in the nucleus. Steps during the processing of the globin mrna.

4 The 5 End of Eukaryotic mrna Is Capped The capping process takes place during transcription and may be important for release from pausing of transcription. The cap blocks the 5 end of mrna and is methylated at several positions. The cap structure is recognized by protein factors to influence mrna stability, splicing, export, and translation.

5 The enzymes responsible for RNA processing ride on the tail of the eucaryotic RNA polymerase as it transcribes an RNA, processing the transcript as it emerges from the RNA polymerase The 3 ends of eukaryotic RNAs are first trimmed by an enzyme that cuts the RNA chain at a particular sequence of nucleotides and are then finished off by a second enzyme that adds a series of repeated adenine (A) nucleotides to the cut end. This poly-a tail is generally a few hundred nucleotides long. 5

6 Nuclear Splice Sites Are Short Sequences Splice sites are the sequences immediately surrounding the exon intron boundaries. They are named for their positions relative to the intron. The 5 splice site at the 5 (left) end of the intron includes the consensus sequence GU. The 3 splice site at the 3 (right) end of the intron includes the consensus sequence AG. G/GU at 5 poly pyrimidin tract..ag/g at 3 Additional conserved sequences at both 5 and 3 splice sites define functional splice sites among numerous other potential sites in the pre-mrna.

7 Splice Sites Are Read in Pairs Splicing junctions are recognized only in the correct pairwise combinations.

8 mrnas Are selectively exported from the Nucleus Poly-A binding proteins, a cap-binding complex, and proteins that mark completed RNA splices exon junction complex (EJC)

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10 An mrna sequence is Decoded in sets of Three Nucleotides - The nucleotide sequence of an mrna is translated into the amino acid sequence ofa protein via the genetic code. - By convention, codons are always written with the 5 -terminal nucleotide to the left. - There are three codons that do not specify any amino acid but act as termination sites (stop codons). - One- codon AUG acts both as an initiation codon, signaling the start of a protein-coding message, and as the codon that specifies methionine. 10

11 Encoding Genetic Information 11

12 The Properties of the Genetic Code A given amino acid tends to be encoded by related codons. Amino acids with similar properties also tend to be clustered Amino acids with acidic side chains are shown in red, those with basic side chains in blue, those with polar in green, and those with hydrophobic side chains in brown This reduces the probability that base substitutions will result in changes in the amino acid sequence of a protein. 12

13 trna Molecules Match Amino Acids to Codons in mrna The translation of mrna into protein depends on adaptor molecules that can recognize and bind to a codon at one site on their surface and to an amino acid at another site. These adaptors consist of a set of small RNA molecules known as transfer RNAs (trnas), each about 80 nucleotides in length. 13

14 Wobble base-pairings Some amino acids have more than one trna and some trnas are constructed so that they require accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch at the third position. Wobble base-pairing explains why so many of the alternative codons for an amino acid differ only in their third nucleotide Wobble base-pairings make it possible to fit the 20 amino acids to their 61 codons. 14

15 Specific enzymes Couple trnas to the Correct Amino Acid trna molecule becomes charged linked to the one amino acid in 20 that is its right partner. Recognition and attachment of the correct amino acid depend on enzymes called aminoacyl-trna synthetases. It covalently couples each amino acid to its appropriate set of trna molecules. There is a different synthetase enzyme for each amino acid. 15

16 One attaches glycine to all trnas that recognize codons for glycine, another attaches phenylalanine to all trnas that recognize codons for phenylalanine and so on. Specific nucleotides in both the anticodon and the amino-acid accepting arm allow the correct trna to be recognized by the synthetas 16

17 Amino Acid Activation Each transfer RNA molecule has to be attached to the correct amino acid. Amino acids are covalently linked to the 3 ends of their cognate trna(s) by an enzyme called an aminoacyl-trna synthetase (aars). Normally, cells contain 20 different aminoacyl-trna synthetases, one for each of the 20 amino acids incorporated into proteins. Any trna whose anticodon recognizes one of the various codons specifying that amino acid will be charged by that aars 17

18 The two ends of the trna the acceptor stem and anticodon are particularly important for recognition by most of these enzymes. A specific base pair in a trna Ala (the G-U base pair involving the third G from the 5 end of the molecule in) is the primary determinant of its interaction with the alanyl-trna synthetase. Insertion of this specific base pair into the acceptor stem of a trnaphe or a trnacys is sufficient to cause these trnas to be recognized by alanyl-trna synthetase and to be aminoacylated with alanine. 18

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20 Aminoacyl-tRNA synthetases carry out the following two-step reaction: 1-) ATP + amino acid aminoacyl-amp + PPi 2-) aminoacyl-amp + trna aminoacyl-trna + AMP it produces a high-energy bond between the charged trna and the amino acid. The energy of this bond is used at a later stage in protein synthesis to link the amino acid covalently to the growing polypeptide chain. Each mrna molecule is eventually degraded into nucleotides by Rnases. 20

21 The RNA Message is Decoded on Ribosomes Ribosome: a large molecular machine that moves along the mrna, captures complementary trna molecules, holds them in position, and covalently links the amino acids that they carry so as to form a protein chain The small subunit matches the trnas to the codons of the mrna. The large subunit catalyzes the formation of the peptide bonds that covalently link the amino acids together into a polypeptide chain. 21

22 The two subunits come together on an mrna molecule, usually near its beginning (5 end), to begin the synthesis of a protein. The mrna is then pulled through the ribosome like a long piece of tape. As the mrna moves through it, the ribosome translates the nucleotide sequence into an amino acid sequence one codon at a time, using the trnas as adaptors. Finally, the two subunits of the ribosome separate when synthesis of the protein is finished 22

23 - Each ribosome contains a binding site for an mrna molecule and three binding sites for trna molecules, called the A-site, the P-site, and the E-site - The appropriate charged trna enters the A-site by base pairing with the complementary codon on the mrna molecule. - Its amino acid is then linked to the peptide chain held by the trna in the neighboring P-site. - Next, the ribosome shifts, and the spent trna is moved to the E-site before being ejected - The chain growing from its amino to its carboxyl end until a stop codon is encountered 23

24 The Ribosome is a Ribozyme Ribosome is composed of two-thirds RNA and one-third protein. rrnas not the proteins are responsible for the ribosome s overall structure and its ability to choreograph protein synthesis. Ribosomal proteins fold and stabilize the RNA core, while permitting the changes in rrna conformation that are necessary for this RNA to catalyze efficient protein synthesis. Three trna-binding sites (the A-, P-, and E-sites) and the catalytic site for peptide bond formation on the ribosome formed primarily by the rrnas. The catalytic site ( is formed by the 23S RNA of the large subunit) precisely orients the two reactants, the elongating peptide and the chargedtrna 24

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26 The translation of an mrna begins with the codon AUG, and a special trna is required to initiate translation. This initiator trna always carries the amino acid methionine. This methionine is usually removed later by a specifi protease. The initiator trna is distinct from the trna that normally carries methionine The initiator trna, coupled to methionine, is first loaded into a small ribosomal subunit, along with additional proteins called translation initiation factors. 26

27 initiation of protein synthesis requires initiation factors and a special initiator trna 27

28 Efficient translation initiation also requires additional proteins that are bound at the 5 cap and poly-a tail of the mrna. Of all the charged trnas in the cell, only the charged initiator trna is capable of binding tightly to the P-site of the small ribosomal subunit 28

29 The end of the protein-coding message is signaled by the presence of one of several codons called stop codons. These special codons UAA, UAG, and UGA are not recognized by a trna and do not specify an amino acid. Proteins known as release factors bind to any stop codon that reaches the A-site on the ribosome, and this binding alters the activity of the peptidyltransferase in the ribosome, causing it to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-trna. The ribosome releases the mrna and dissociates into its two separate subunits, which can then assemble on another mrna molecule to begin a new round of protein synthesis 29

30 Newly synthesized proteins are typically met by their chaperones as they emerge from the ribosome 30

31 Proteins Are Made on polyribosomes. Inhibitors of procaryotic protein synthesis are used as antibiotics Many common antibiotics were fist isolated from fungi 31

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