Chapter 17 From Gene to Protein PDF

**Chapter 17** ============== FROM GENE TO PROTEIN ==================== DNA is the instructions (program) that tells the cell what to do. Proteins are the results of those instructions. **Molecular Genetics: The Protein vs. DNA Debate** \>\>\>\>\>\>The study of metabolic defects provided evidenc...

**Chapter 17** ============== FROM GENE TO PROTEIN ==================== DNA is the instructions (program) that tells the cell what to do. Proteins are the results of those instructions. **Molecular Genetics: The Protein vs. DNA Debate** \>\>\>\>\>\>The study of metabolic defects provided evidence that genes lead to proteins. Archibald Garrod first proposed this relationship in 1909 -suggested that genes dictated phenotypes via enzymes -inherited diseases were the result of the lack of an enzyme \>\>\>How genes control metabolism. The suggestion of Garrod were confirmed in the 1930\'s George Beadle and Edward Tatum conducted experiment with yeast, *Neurospora crassa,* and demonstrated that different mutants (generated by X-rays) had the pathway of arginine (an amino acid) synthesis blocked at different steps.(see fig 17.2) ----------------- --------------------- ---------------- ----------------- --------------- Minimal medium (MM) MM + ornithine MM + citrulline MM + arginine Wild type \+ \+ \+ \+ Mutant type I \- \+ \+ \+ Mutant type II \- \- \+ \+ Mutant type III \- \- \- \+ ----------------- --------------------- ---------------- ----------------- --------------- \+ = growth, - = no growth Wild-type yeast could survive on minimal medium. The mutants were **auxotrophs** that required added nutrients. \-\--**Auxotrophs** Beadle and Tatum deduced that the 3 different mutant types each lacked a different enzyme in the pathway that synthesizes arginine. From these results they formulated the **one gene-one enzyme** hypothesis This has since been modified to the **one gene-one polypeptide** hypothesis because: -Not all proteins are enzymes -Many enzymes are comprised of 2 polypeptide subunits \>\>\>\>\>\>The steps from DNA to protein (see fig 17.4). DNA ==\> RNA ==\> Proteins \>\>\>Step one - DNA to RNA \-\--**Transcription** -RNA that is synthesized from a gene coding for a protein is called DNA verses RNA -Similar because they are both polymers of nucleotides -Structural differences -sugar - deoxyribose (DNA) verses ribose (RNA) -base - thymine (DNA) is replaced by uracil (RNA) \>\>\>Step two - RNA to protein This process is called **translation**. \-\--**Translation** Why translation?? Different language of nucleic acids and proteins: 4 bases in nucleic acids 20 amino acids in proteins Translation occurs on the ribosomes In prokaryotes, since there is no nucleus, transcription and translation occur in rapid succession. In eukaryotes the two processes are separated in time and space. -Transcription occurs in the nucleus -The mRNA that is made must be modified before moving into the \>\>\>\>\>\>Transcription of DNA to RNA (see fig. 17.8) Like DNA replication, transcription of a DNA sequence to mRNA for a protein occurs by building the new molecule in a 5\' =\> 3\' direction. This means reading the template in the 3\' =\> 5\' direction. During transcription of a gene, only one strand (template strand) of the DNA's two strands is read. Different genes use different strands as the template strand. The enzyme RNA polymerase catalyzes transcription. This enzyme: 1\) Separates the DNA helix at a specific sequence The initiation site + gene + terminator make up the **transcription unit** \-\--**Transcription unit** \>\>\>Initiation of transcription (see fig. 17.9) RNA polymerase binds to the DNA at the **promoter** region of the gene. \-\--**Promoter** In eukaryotes, RNA polymerase cannot recognize the promoter without the help of **transcription factors** \-\--**Transcription factors** The transcription of eukaryote mRNA by RNA polymerase usually requires a specific transcription factor that binds to a DNA region known as a **TATA box**. \-\--**TATA box** The RNA polymerase recognizes the TATA transcription factor-DNA complex and binds the DNA. Other transcription factors or initiation factors may bind before transcription begins. The active RNA polymerase separates the two DNA strands at the initiation site and transcription begins. \>\>\>Elongation of the RNA strand Once transcription begins RNA polymerase performs two functions: 1\. Untwisting the DNA double helix for about ten nucleotides to expose 2\. Catalyze the linkage of new RNA nucleotide to the 3\' end of the RNA Elongation of mRNA occurs at about 30 - 60 nucleotides per second. As elongation proceeds: Several sequential RNA transcripts can be generated from a single gene. (i.e., as one transcript is being initiated others may be at various stages of elongation) \>\>\>Termination of transcription At the end of the DNA transcription unit for a gene there is a **terminator**. \-\--**Terminator** \>\>\>\>\>\>Eukaryotic cells modify RNA after transcription Before eukaryotic mRNA is exported from the nucleus it is processed in two ways: 1. Both ends are covalently altered (see fig. 17.11) 2\) Intervening sequences (**introns**) are removed and the remaining \>\>\>Modification of the mRNA ends During mRNA processing both the 5\' and 3\' ends are modified. \-\--**5\' cap** -acts with the leader sequence to bind the mRNA to ribosomes. -The 3\' end of the mRNA has a **poly-A tail** added \-\--**Poly-A tail** -aids in export of mRNA from the nucleus -attached to a trailer sequence at the end of the mRNA \>\>\>**RNA splicing** In eukaryote the original RNA transcript is the complement of the DNA sequence for the gene, however the functional mRNA in the cytoplasm is much shorter. Between transcription and translation the RNA is processed to remove parts of the sequence. The DNA and the complementary pre-mRNA consists of the coding sequence interrupted by noncoding segments, called intervening sequences or **introns**. \-\--**Introns** The coding sequences of the DNA and pre-mRNA are known as **exons** because they are the sequence that is expressed as proteins. \-\--**Exons** The exon regions often code for different **domains** of a protein (see fig.17.14) \-\--**domains** Because these domains are on separate exons the opportunity for a function to be swapped from one protein to another is greater. RNA splicing, as part of the RNA processing that occurs before the mRNA leaves the nucleus, removes the introns from the hnRNA \-\--**RNA splicing** -RNA splicing also occurs during post-transcriptional modification of tRNA and rRNA. RNA splicing involves enzymes and other protein factors. In addition, small nuclear ribonucleoproteins (snRNPs) play a key role. Several snRNPs along with other proteins assemble to make a **spliceosome (see fig. 17.13)**. \-\--**Spliceosome** The spliceosome brings together the exons and excises the intron. The exons are joined and the excised intron is released as a lariat-shaped loop. Differential splicing (removal of all versus some introns) can result in to different mRNA products from a single pre-mRNA transcript. \>\>\>\>\>\>Translation is the RNA-directed synthesis of polypeptides (see fig. 17.15). During translation, proteins are synthesized according to the genetic message of sequential **codons** in the mRNA. \-\--**Codon** -In part of this role as "interpreter", the tRNA must "read" the mRNA. This is accomplished by the **anticodon** portion of the tRNA (see fig 17.16) \-\--**Anticodon** -The other portion the tRNA's role as "interpreter" is to transfer the correct amino acid from the cytoplasmic pool of amino acids to the ribosome for protein The specificity of this correct pairing is accomplished by a group of enzymes known as **aminoacyl-tRNA synthetases (see fig. 17.17)** \-\--**Aminoacyl-tRNA synthetases** This is a two-step process 1. **Activation of the amino acid** This process occurs before the anticodon pairs up with the codon on the mRNA Using this one codon =\> one anticodon =\> one amino acid method the gene is decoded to protein. \>\>\>The **ribosome** is where proteins are built.(see fig 17.18) The ribosome coordinates the pairing of tRNA anticodons with mRNA codons. Eukaryotic ribosome structure -Two subunits (large and small) -Composed of 60% **ribosomal RNA** (**rRNA**) and 40% protein -The ribosomal subunits are made in the nucleolus. -The subunits combine as a ribosome only when they are translating a protein In addition to the mRNA binding site (the groove between subunits), the ribosome also has 3 tRNA binding sites (P, A, and E) The (peptidyl-tRNA binding) **P site** -holds the tRNA with the polypeptide chain attached. The (aminoacyl-tRNA binding) **A site** -hold the aminoacyl-tRNA with the next amino acid to be added. The (tRNA discharge or exit) **E site** -releases the tRNA once the peptide chain has been transferred to the The ribosome holds all the components together as the next amino acid is transferred to the growing polypeptide chain. \>\>\>\>\>\>In the genetic code, a triplet of nucleotides specifies an amino acid Dictated by the math since there are 4 nucleotides and 20 amino acids. -if it was a 1:1 relationship then only 4 amino acids would be needed -a 2:1 would result in 16 possible amino acids (4^2^). -as a 3:1 there could be as many as 64 amino acids (4^3^) So a triplet of nucleotides is the smallest size that could code for all the amino acids. \>\>\>The genetic \"code\" (see fig 17.6) 61 of the 64 possible triplet code for amino acids. The remaining three triplets signal the translation to stop. Since there are only 20 amino acids, more than one triplet can code for the same amino acid. This relationship is known as redundancy and usually the codons differ only at the third position. There is no ambiguity in the triplet code since a given triplet codes for one and only one amino acid. The correct ordering and grouping of nucleotide is an important aspect of the translation of the triplet codons. The correct ordering is the **reading frame**. \-\--**Reading frame** The genetic "code" is nearly universal -The genes in the mitochondria and the chloroplast can vary. The first two positions of the anticodon compliment the codon exactly, the third position allows **wobble** in the base-pairing. \-\--**Wobble** Because of wobble only 45 different tRNAs are needed to complement the 64 possible codons. \>\>\>Building a polypeptide. Protein synthesis occurs in three stages: 2. **Elongation** Initiation of translation (see fig 17.19) 1\. Assembly of mRNA, initiator tRNA (with methionine), and small ribosome subunit. Requires protein initiation factors 2\. Large ribosome subunit joins complex, and the initiator tRNA is in the P site The elongation cycle of translation (see fig 17.20) 1\. The next tRNA occupies the A site and the anticodon hydrogen bonds to the codon of the mRNA. -energy for this step is provided by the hydrolysis of GTP=\> GDP + P -require a protein elongation factor 2\. Peptide bond formation -this reaction as catalyzed by a **ribozyme** -this leaves the tRNA at the P site with no amino acid, and the tRNA at the 3\. Translocation -the "empty" tRNA moves to the E site and leaves -the mRNA with the attached tRNA moves through ribosome in 5\' =\> 3\' -energy for this step is provided by the hydrolysis of GTP=\> GDP + P -elongation proceeds at \~1 million amino acids per minute. The termination of translation (see fig 17.21) 1\. The mRNA reaches a stop codon: UAA, UAG, or UGA 2\. A release factor binds to the A site 3\. The peptidyl transferase adds H~2~O instead of an amino acid to the end of -this frees polypeptide from tRNA in P site \>\>\>**Polyribosomes** can quickly make many copies of a protein from a single mRNA (see fig 17.23) \-\--**Polyribosomes** and begin translation. -Several ribosomes may translate an mRNA at once, making many copies of a polypeptide. \>\>\>Proteins are targeted for specific destinations by signal peptides (see fig 17.22) If a protein is to be inserted into a membrane (integral proteins) or to be shipped to a specific compartment (or out of the cell), then it is directed to the correct location by a **signal peptide**. The signal peptide combines with a **signal recognition particle** (**SRP**) to direct the protein to the proper location. \-\--**signal peptide** \-\--**signal recognition particle (SRP)** \>\>\>Coupled transcription-translation in prokaryotes (see fig. 17.24) In prokaryotes the mRNA may be translated as soon as the initiation codon is transcribed. -No nucleus to separate the new mRNA from the ribosomes. -No mRNA processing (splicing or modification) needed in prokaryotes \>\>\>\>\>\>**Point mutations** can affect the function of a protein (see fig 17.26). \-\--**Point mutations** \>\>\>Types of point mutations(see fig. 17.27) \-\--**Substitutions** -Change may be drastic Base-pair substitution mutations are usually **missense mutations** or **nonsense mutations**. \-\--**Missense mutations** \-\--**Nonsense mutations** **Insertion** or **Deletion**: can result in a **frameshift mutation**. \-\--**Insertion** \-\--**Deletion** \-\--**Frameshift mutations** \>\>\>**Mutagenesis** may be spontaneous or more often is the result of **mutagens**. \-\--**Mutagens** Regardless of the cause of mutations the most common phenotypic effect is cancerous cell growth.