DNA Transcription and Translation in the Cell, Lecture notes of Cell Biology

Download DNA Transcription and Translation in the Cell and more Lecture notes Cell Biology in PDF only on Docsity! CYTOGENETICS Lesson 11 [TRANS] LESSON 11: DNA TRANSCRIPTION & PROTEIN TRANSLATION CENTRAL DOGMA REPLICATION ● DNA is copied and passed on to daughter cells ● All cells, from bacteria to those in humans, express their genetic information in this way—a principle so fundamental that it has been termed the central dogma of molecular biology. ● “Genetic information directs the synthesis of proteins. The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells. DNA can also be copied—or replicated—to produce new DNA molecules. The segments of DNA that are transcribed into RNA are called genes.” TRANSCRIPTION ● DNA is converted into RNA ● The first step in gene expression, the process by which cells read out the instructions in their genes, is transcription. ● “The process is called transcription because the information, though copied into another chemical form, is still written in essentially the same language— the language of nucleotides.” TRANSLATION ● RNA is read and converted into proteins ● “For most genes, RNA serves solely as an intermediary on the pathway to making a protein. For these genes, each RNA molecule can direct the synthesis, or translation, of many identical protein molecules. This successive amplification enables cells to rapidly synthesize large amounts of protein whenever necessary. At the same time, each gene can be transcribed, and its RNA translated, at different rates, providing the cell with a way to make vast quantities of some pro- teins and tiny quantities of others.” GENE EXPRESSION GENE EXPRESSION ● Sequences of DNA that can be converted into products, either proteins or RNA ● Genes expression rates can be highly variable ● “A cell can express different genes at different rates. In this and later figures, the portions of the DNA that are not transcribed are shown in gray.” RNA VS. DNA RNA ● Phosphodiester bonds ● Base pairing ● Single stranded ● Sugar: ribose ● Uracil ○ Complementary to Adenine ● Can adopt a variety of forms ○ Conventional base pairing: ■ A and U, C and G DNA ● Phosphodiester bonds ● Base pairing ● Double stranded ● Sugar: deoxyribose ● Thymine ○ Complementary to Adenine 1 LESSON 11: DNA TRANSCRIPTION & PROTEIN TRANSLATION RNA TRANSCRIPTION ● Carried out by RNA polymerase ○ Unwinds DNA ○ Attaches ribonucleoside triphosphates (ATP, CP, UTP, and GTP) ○ Does not need a primer ○ Cannot perform proofreading ● A new RNA strand can be synthesized before the first RNA has been completed ○ Rapid creation of RNA transcripts ● “DNA is transcribed into RNA by the enzyme RNA polymerase. (A) RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix in front of it. As it progresses, the polymerase adds ribonucleotides one-by- one to the RNA chain, using an exposed DNA strand as a template. The resulting RNA transcript is thus single-stranded and complementary to the template strand TYPES OF RNA TRANSCRIPTS ● Only mRNAs are translated into proteins SIGNALS FOR TRANSCRIPTION ● Transcription start site - the beginning site of the gene ● Promoter - nucleotides directly upstream of the start site ○ Attachment site for RNA polymerase ● Terminator (stop site) - causes polymerase stop and release both the DNA template and the newly made RNA transcript ● “When an RNA polymerase collides randomly with a DNA molecule, the enzyme sticks weakly to the double helix and then slides rapidly along its length. RNA polymerase latches on tightly only after it has encountered a gene region called a promoter, which contains a specific sequence of nucleotides that lies immediately upstream of the starting point for RNA synthesis. As it binds tightly to this sequence, the RNA polymerase opens up the double helix immediately in front of the promoter to expose the nucleotides on each strand of a short stretch of DNA. One of the two exposed DNA strands then acts as a template for complementary base-pairing with incoming ribonucleoside triphosphates, two of which are joined together by the polymerase to begin synthesis of the RNA strand. Elongation then continues until the enzyme encounters a second signal in the DNA, the terminator (or stop site), where the polymerase halts and releases both the DNA template and the newly made RNA transcript. The terminator sequence itself is also transcribed, and it is the interaction of this 3ʹ segment of RNA with the polymerase that causes the enzyme to let go of the template DNA. TYPES OF RNA POLYMERASE ● RNA polymerase Il transcribes mRVA for protein synthesis GENERAL TRANSCRIPTION FACTORS ● Accessory proteins needed by RNA polymerase Il to begin transcription ● TFIID - first to bind to the promoter ● landmark for the assembly of other TF proteins ● Binds to TATA box ● Composed of T and A nucleotides ● ~30 bp before the start site ● Other factors bind to form the complete transcription initiation complex ● RNA polymerase is released ● Addition of phosphates byТРIIН ● Other factors bind to form the complete transcription initiation complex ● RNA polymerase is released ● Addition of phosphates by TFIIH ● Most TFs are released once transcription begins ● “To begin transcription, eukaryotic RNA polymerase II requires a set of general transcription factors. These factors are designated TFIIB, TFIID, and so on. TFIIH also phosphorylates RNA polymerase II, releasing the polymerase from most of the general transcription factors, so it can 2 LESSON 11: DNA TRANSCRIPTION & PROTEIN TRANSLATION PROTEIN TRANSLATION TRANSLATION ● mRNA are read in groups of 3 nucleotides - codon ○ Translated into amino acids using the genetic code ○ 64 codons ○ Some codons are redundant ● “The nucleotide sequence of an mRNA is translated into the amino acid sequence of a protein via the genetic code. All of the three-nucleotide codons in mRNAs that specify a given amino acid are listed above that amino acid, which is given in both its three-letter and one-letter abbreviations.” TRANSFER RNAS ● RNAs that carry amino acids and detect codons in the mRNA ○ 3' end - carries the amino acid ○ Anticodon - set of 3 nucleotides that are complementary to the codon ● Mismatch or wobble is possible in the 3rd position ● 31 kinds of tRNA molecules ● “tRNA molecules are molecular adaptors, linking amino acids to codons. In this series of diagrams, the same tRNA molecule—in this case, a tRNA specific for the amino acid phenylalanine (Phe)—is depicted in various ways. “ AMINOACYL-TRNA SYNTHETASES ● Attach the amino acid to the corresponding IRNA ● 20 aminoacyl-tRNA synthetases ● “The genetic code is translated by aminoacyl-tRNA synthetases and tRNAs. Each synthetase couples a particular amino acid to its corresponding tRNAs, a process called charging. The anticodon on the charged tRNA molecule then forms base pairs with the appropriate codon on the mRNA. An error in either the charging step or the binding of the charged tRNA to its codon will cause the wrong amino acid to be incorporated into a polypeptide chain. In the sequence of events shown, the amino acid tryptophan (Trp) is specified by the codon UGG on the mRNA. RIBOSOMES ● Brings together the mRNA and tRNA ● Used to form the amino acid chai n ○ Ribosomal RNA (rRN A) ■ Catalyze reaction ○ Ribosomal protei n ■ Stabilize the rRNA ● Small subunit ○ matches the tRNAs to , the codons of the mRNA ● Large subunit ○ Peptidyl transferase activity ● Catalyzes peptide bonds to lengthen AA chain ● “The eukaryotic ribosome is a large complex of four rRNAs and more than 80 small proteins. Prokaryotic ribosomes are very similar: both are formed from a large and small subunit, which only come together after the small subunit has bound an mRNA. The RNAs account for most of the mass of the ribosome and give it its overall shape and structure.” RIBOSOME BUILDING SITES ● A site - aminoacyl-tRNA 5 LESSON 11: DNA TRANSCRIPTION & PROTEIN TRANSLATION ○ Where tRNA enters ● P site - peptidyl-tRNA ○ Holds the polypeptide chain ● E site - exit ○ Where tRNA exits ● “Each ribosome has a binding site for an mRNA molecule and three binding sites for tRNAs. The tRNA sites are designated the A, P, and E sites (short for aminoacyl-tRNA, peptidyl-tRNA, and exit, respectively).” TRANSLATION ON THE RIBOSOME ● Translation takes place in a four-step cycle, which is repeated over and over during the synthesis of a protein. In step 1, a charged tRNA carrying the next amino acid to be added to the polypeptide chain binds to the vacant A site on the ribosome by forming base pairs with the mRNA codon that is exposed there. ● Only a matching tRNA molecule can base-pair with this codon, which determines the specific amino acid added. The A and P sites are sufficiently close together that their two tRNA molecules are forced to form base pairs with codons that are contiguous, with no stray bases in-between. This positioning of the tRNAs ensures that the correct reading frame will be preserved throughout the synthesis of the protein. In step 2, the carboxyl end of the polypeptide chain (amino acid 3 in step 1) is uncoupled from the tRNA at the P site and joined by a peptide bond to the free amino group of the amino acid linked to the tRNA at the A site. This reaction is carried out by a catalytic site in the large subunit. In step 3, a shift of the large subunit relative to the small subunit moves the two bound tRNAs into the E and P sites of the large subunit. In step 4, the small subunit moves exactly three nucleotides along the mRNA molecule, bringing it back to its original position relative to the large subunit. This movement ejects the spent tRNA and resets the ribosome with an empty A site so that the next charged tRNA molecule can bind.” SETTING THE READING FRAME ● Starting at the correct nucleotide is essential to create the correct protein ● Start codon ○ AUG - carries methionine TRANSLATION INITIATION ● Initial complex that forms: ○ Small ribosomal subunit ○ Translation initiation factors ○ Initiator tRNA - carries methionine ● Binds to the P site ● Binds to 5' end of mRNA ● Small ribosomal subunit locates the start codon - AUG ○ Signifies the starting amino acid ○ Ensures that the reading frame is correct 6 LESSON 11: DNA TRANSCRIPTION & PROTEIN TRANSLATION ● Large ribosomal subunit attaches ● Translation begins ● “Initiation of protein synthesis in eukaryotes requires translation initiation factors and a special initiator tRNA. Although not shown here, efficient translation initiation also requires additional proteins that are bound at the 5ʹ cap and poly-A tail of the mRNA In this way, the translation apparatus can ascertain that both ends of the mRNA are intact before initiating translation. Following initiation, the protein is elongated by the reactions outlined” TRANSLATION TERMINATION ● Stop codons - UAA, UAG, and UGA ○ No corresponding tRNA ○ Release factor proteins attach instead ● Catalyzes the addition of a water molecule instead of an amino acid in the P site. ● AA chain is released ● Ribosome dissociates EUKARYOTIC PROTEIN TRANSLATION ● Eukaryotic mRNA is monocistronic ○ 1 mRNA codes for only 1 protein ● Multiple ribosomes can translate mRNA at the same time ○ Polyribosomes or polysomes ● “Proteins are synthesized on polyribosomes. (A) Schematic drawing showing how a series of ribosomes can simultaneously translate the same mRNA molecule. (B) Electron micrograph of a polyribosome in the cytosol of a eukaryotic cell.” TAKEAWAYS ● The flow of genetic information in all living cells is DNA → RNA → protein. The conversion of the genetic instructions in DNA into RNAs and proteins is termed gene expression. ● To express the genetic information carried in DNA, the nucleotide sequence of a gene is first transcribed into RNA. Transcription is catalyzed by the enzyme RNA polymerase, which uses nucleotide sequences in the DNA molecule to determine which strand to use as a template, and where to start and stop transcribing. ● RNA differs in several respects from DNA. It contains the sugar ribose instead of deoxyribose and the base uracil (U) instead of thymine (T). RNAs in cells are synthesized as single-stranded molecules, which often fold up into complex three-dimensional shapes. ● Cells make several functional types of RNAs, including messenger RNAs (mRNAs), which carry the instructions for making proteins; ribosomal RNAs (rRNAs), which are the crucial components of ribosomes; and transfer RNAs (tRNAs), which act as adaptor molecules in protein synthesis. ● To begin transcription, RNA polymerase binds to specific DNA sites called promoters that lie immediately upstream of genes. To initiate transcription, eukaryotic RNA polymerases require the assembly of a complex of general 7