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CYTOGENETICS
Lesson
[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
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
● “ Eukaryotic mRNA molecules are modified by capping and polyadenylation. (A) A eukaryotic mRNA has a cap at the 5ʹ end and a poly-A tail at the 3 ʹ end. In addition to the nucleotide sequences that code for protein, most mRNAs also contain extra, noncoding sequences, as shown. The noncoding portion at the 5 ʹ end is called the 5ʹ untranslated region, or 5ʹ UTR, and that at the 3ʹ end is called the 3 ʹ UTR. (B) The structure of the 5 ʹ cap. Many eukaryotic mRNA caps carry an additional modification: the 2 ʹ-hydroxyl group on the second ribose sugar in the mRNA is methylated.” ● Introns - long, noncoding, intervening sequences ● Exons - coding sequences ○ Typically shorter than introns ● Pre-mRNA or precursor-mRNA ○ Contains broth introns and exons ● “ Eukaryotic and bacterial genes are organized differently. A bacterial gene consists of a single stretch of uninterrupted nucleotide sequence that encodes the amino acid sequence of a protein. In contrast, the protein-coding sequences of most eukaryotic genes (exons) are interrupted by noncoding sequences (introns). Promoter sequences are indicated in green.” ● RNA Splicing ○ Removal of introns from RNA ○ Detected by specific recognition sequences ● Done by the spliceosome ● small nuclear ribonucleoproteins (snRNPs) ● Proteins and small nuclear RNAs (snRNAs) ● “ An intron in a pre-mRNA molecule forms a branched structure during RNA splicing. In the first step, the branch-point adenine (red A) in the intron sequence attacks the 5 ʹ splice site and cuts the sugar–phosphate backbone of the RNA at this point” ALTERNATIVE SPLICING ● Allows for multiple genes to be produced from a single gene ● “ Some pre-mRNAs undergo alternative RNA splicing to produce different mRNAs and proteins from the same gene. Whereas all exons are transcribed, they can be skipped over by the spliceosome to produce alternatively spliced mRNAs, as shown. Such skipping occurs when the splicing signals at the 5ʹ end of one intron are paired up with the branch-point and 3ʹ end of a different intron. An important feature of alternative splicing is that exons can be skipped or included; however, their order—which is specified in the DNA sequence—cannot be rearranged.“ CYTOPLASMIC EXPORT OF MRNA ● Must contain: ○ poly-A-binding proteins ; cap-binding complex ○ proteins that bind to mRNAs that have been appropriately spliced ● mRNA is degraded shortly after release into the cytoplasm ● “ A specialized set of RNA-binding proteins signals that a completed mRNA is ready for export to the cytosol. As indicated on the left, the 5’ cap and poly-A tail of a mature mRNA molecule are “marked” by proteins that recognize these modifications. Successful splices are marked by exon junction complexes”
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 w obble 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 chain ○ Ribosomal RNA (rRNA) ■ Catalyze reaction ○ Ribosomal protein ■ 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
● 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
transcription factors at the promoter, whereas bacterial RNA polymerase requires only an additional subunit, called sigma factor. ● Most protein-coding genes in eukaryotic cells are composed of a number of coding regions, called exons, interspersed with larger, noncoding regions, called introns. When a eukaryotic gene is tran- scribed from DNA into RNA, both the exons and introns are copied. ● Introns are removed from the RNA transcripts in the nucleus by RNA splicing, a reaction catalyzed by small ribonucleoprotein complexes known as snRNPs. Splicing removes the introns from the RNA and joins together the exons—often in a variety of combinations, allowing multiple proteins to be produced from the same gene. ● Eukaryotic pre-mRNAs go through several additional RNA process- ing steps before they leave the nucleus as mRNAs, including 5 ʹ RNA capping and 3 ʹ polyadenylation. These reactions, along with splicing, take place as the pre-mRNA is being transcribed. ● Translation of the nucleotide sequence of an mRNA into a protein takes place in the cytoplasm on large ribonucleoprotein assemblies called ribosomes. As the mRNA moves through the ribosome, its message is translated into protein. ● The nucleotide sequence in mRNA is read in consecutive sets of three nucleotides called codons; each codon corresponds to one amino acid. ● The correspondence between amino acids and codons is specified by the genetic code. The possible combinations of the 4 different nucleotides in RNA give 64 different codons in the genetic code. Most amino acids are specified by more than one codon. ● tRNAs act as adaptor molecules in protein synthesis. Enzymes called aminoacyl-tRNA synthetases covalently link amino acids to their appropriate tRNAs. Each tRNA contains a sequence of three nucleo- tides, the anticodon, which recognizes a codon in an mRNA through complementary base-pairing. ● Protein synthesis begins when a ribosome assembles at an initia- tion codon (AUG) in an mRNA molecule, a process that depends on proteins called translation initiation factors. The completed protein chain is released from the ribosome when a stop codon (UAA, UAG, or UGA) in the mRNA is reached. ● The stepwise linking of amino acids into a polypeptide chain is catalyzed by an rRNA molecule in the large ribosomal subunit, which thus acts as a ribozyme. ● The concentration of a protein in a cell depends on the rates at which the mRNA and protein are synthesized and degraded. Protein degradation in the cytosol and nucleus occurs inside large protein complexes called proteasomes. ● From our knowledge of present-day organisms and the molecules they contain, it seems likely that life on Earth began with the evolu- tion of RNA molecules that could catalyze their own replication. ● It has been proposed that RNA served as both the genome and the catalysts in the first cells, before DNA replaced RNA as a more stable molecule for storing genetic information, and proteins replaced RNAs as the major catalytic and structural components. RNA catalysts in modern cells are thought to provide a glimpse into an ancient, RNA- based world.
