Protein Synthesis PDF

Protein Synthesis From Gene to Protein  History Archibald Garrod – 1909 First to suggest that genes dictate phenotype through production of proteins Believed that genetic diseases resulted from the inability to make particular enzymes “Inborn errors of metabolism” ...

Protein Synthesis From Gene to Protein  History Archibald Garrod – 1909 First to suggest that genes dictate phenotype through production of proteins Believed that genetic diseases resulted from the inability to make particular enzymes “Inborn errors of metabolism”  One Gene – One Polypeptide Not all proteins are enzymes Can extend one gene = one enzyme doctrine to one gene = one polypeptide Many proteins are comprised of two or more polypeptides  Central Dogma How does the sequence of a strand of DNA correspond to the amino acid sequence of a protein? The central dogma of molecular biology, states that:  DNA is kept in the nucleus for the protection of the genetic code mRNA makes a copy of the DNA and leaves the nucleus through the nuclear envelope Once the mRNA reaches the cytoplasm, ribosomes translate the code into a polypeptide which folds into a working protein  What is RNA? RNA has the same primary structure as DNA – consists of a sugar-phosphate backbone, with nucleotides attached to the 1' C of the sugar. Differences between DNA and RNA : – RNA contains the sugar ribose instead of deoxyribose – The nucleotide, uracil, is substituted for thymine – RNA exists as a single-stranded molecule. Because of the extra hydroxyl group on the sugar, RNA is too bulky to form a a stable  Types of RNA mRNA - messenger RNA – A copy of a gene. – Has a sequence complementary to one strand of the DNA & identical to the other strand. – Carries the information stored in DNA in the nucleus to the ribosomes in the cytoplasm where protein is made. tRNA - transfer RNA – A small RNA with a very specific structure that can bind an amino acid at one end, and mRNA at the other end. – Acts as an ‘adaptor’ to carry & attach amino acids to the appropriate place on the  Types of RNA (Cont.) rRNA - ribosomal RNA – One of the structural components of the ribosome. – Has a sequence complimentary to regions of the mRNA – Allows ribosomes to bind to an mRNA snRNA - small nuclear RNA – Is involved in the machinery that processes RNA's as they travel between the nucleus and the cytoplasm.  The Genetic Code How does mRNA specify an amino acid sequence? It would be impossible for each amino acid to be specified by one nucleotide – there are only 4 nucleotides and 20 amino acids. – two nucleotide combinations could only specify 16 amino acids. Each amino acid is specified by a combination of three nucleotides, called a codon  The Code is Redundant, Not Ambiguous Each amino acid may be specified by up to six codons – In many cases, codons that are synonyms differ only in the third base of the triplet Different organisms have different frequencies of codon usage. – A giraffe might use CGC for arginine much more often than CGA, and the reverse might be true for a sperm whale. Some codons specify “stop” or “start There is no ambiguity – the same codon ALWAYS codes for the same amino acid  Transcription The process in which DNA is used as a template for the production of complementary mRNA which is then free to move to the cytoplasm. Highly similar to DNA replication. Different enzymes are used in transcription. The most important is RNA polymerase  RNA Polymerase RNA polymerase is a holoenzyme – an agglomeration of many different factors Together, direct the synthesis of mRNA Pries the DNA strands apart Strings complimentary RNA nucleotides on the DNA template Like DNA polymerase, can only add to the 3’ end So only one mRNA is made, elongating 5’  3’  Stages of Transcription Initiation Elongation Termination  Initiation RNA polymerase must recognize the beginning of a gene to know where to start synthesizing mRNA. One part of the enzyme has a high affinity for a particular DNA sequence that appears at the beginning of genes. The sequence where RNA polymerase attaches to the DNA and begins transcription = the promoter – Tells RNA polymerase both where to start and in which direction or on which strand to continue synthesis.  Eukaryotic Promoters In eukaryotes special proteins, transcription factors, mediate binding RNA polymerase and the promoter RNA polymerase binds to the promoter only after transcription factors bind Transcription factors + RNA polymerase, bound to the promoter = transcription initiation complex Eukaryotic promoters usually include a TATA box – A nucleotide sequence containing TATA about 25 nucleotides prior to the start  Elongation The RNA polymerase stretches open the double helix at the start point in the DNA and begins synthesis of a complementary RNA strand on one of the DNA strands The RNA polymerase recruits RNA nucleotides in the same way that DNA polymerase recruits DNA nucleotides. Since synthesis only proceeds in the 5' to 3' direction, there is no need for Okazaki fragments.  Sense & Antisense Synthesis only occurs in the 5’ to 3’ direction In transcription, only one DNA strand is copied The strand that is copied is called the antisense or template strand The other strand, which is identical to the mRNA made (substituting U for T), is the sense or coding strand.  Termination of Transcription How does RNA polymerase know when to stop transcribing a gene? Sequence that signals the end of transcription = terminator RNA polymerase transcribes the terminator – The transcribed terminator actually ends the process The terminator sequence of the mRNA allows it to form a hairpin loop, which blocks the polymerase. – RNA polymerase falls off the DNA and  Post Transcription Modification In eukaryotes, enzymes modify pre-mRNA before it is sent to the cytoplasm Both ends of the transcript are altered The 5’ end is capped with 7 modified guanines – Protects mRNA from degradation – Helps attach the ribosome At the 3’ end the enzyme poly-A polymerase makes a poly-A tail formed from 50 to 250 adenine nucleotides – Inhibits degradation and helps ribosome attach – May also help export mRNA out of the nucleus Within the interior of the mRNA sections are cut out and the remaining parts are spliced RNA Processing  Introns & Exons Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides = introns Noncoding sequences are interspersed between coding sections Coding sections = exons RNA polymerase transcribes both introns and exons  RNA Splicing Introns are cut out and exons are spliced together before mRNA exits the nucleus Short nucleotide sequences at the end of introns are signals for RNA splicing Small nuclear ribonucleoproteins (snRNPs) recognize splice sites – Composed of snRNA & protein Several snRNPs and additional proteins form a complex = spliceosome The Spliceosome  Translation The process by which ribosomes assemble amino acids in a specific sequence to make polypeptides (proteins) using the information encoded in the mRNA mRNA travels to ribosomes in the cytoplasm, where the message is read The specified amino acids are assembled on the mRNA template on the ribosome  The tRNA An 80 nucleotide RNA sequence that folds into a cloverleaf structure The bottom arm of the tRNA consists of an anti- codon triplet that is complementary to the mRNA sequence The open arm binds to the appropriate amino acid that the tRNA has been designated to carry Each tRNA is specific to only one amino acid The amino acid attachment is catalyzed by 20 different aminoacyl-tRNA synthetase enzymes  The Ribosome The cellular factory where proteins are synthesized Consists of structural RNA and ~ 80 different proteins. In its inactive state, it exists as two subunits – a large subunit and a small subunit. When the small subunit encounters an mRNA, it begins translation of the mRNA to protein. There are three sites in the large subunit – The A site accepts a new tRNA bearing an amino acid – the P site bears the tRNA attached to the  Initiation of Translation The start signal for translation is the codon AUG – Codes for methionine. – Not every protein starts with methionine, – Often this first amino acid will be removed in post-translational processing. A tRNA charged with methionine binds to the translation start signal. The large subunit binds to the mRNA and the small subunit Elongation begins.  Elongation of the New Protein After the first charged tRNA appears in the A site, the ribosome shifts so that the tRNA is in the P site. A new charged tRNA, corresponding to the codon of the mRNA, enters the A site, and a peptide bond is formed between the two amino acids by peptidyl transferase. The first tRNA is now released As the ribosome shifts again to the next codon, the released tRNA moves into the E site where it then moves away from the ribosome At the same time, the tRNA carrying two amino acids moves into the P site A new charged tRNA can bind to the A site. This process of elongation continues until the ribosome reaches a stop codon. Termination of the Protein When the ribosome reaches a stop codon, a release factor binds to the empty A site. This is the ribosome’s signal to break into its large and small subunits, Releasing the new protein and the mRNA.