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4-11-17 Bell Ringer. The base sequence of a fragment DNA is: GAC TCC GTA ATC AAA TGC What is the base sequence on the mRNA molecule transcribed from it? What are the 3 steps of transcription?. Transcription and Translation. Ribosome: Large and Small subunit. 7.2 Understandings:
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4-11-17Bell Ringer • The base sequence of a fragment DNA is:GAC TCC GTA ATC AAA TGCWhat is the base sequence on the mRNA molecule transcribed from it? • What are the 3 steps of transcription?
Transcription and Translation Ribosome: Large and Small subunit
7.2 Understandings: • Transcription occurs in a 5’ to 3’ direction. • Nucleosomes help to regulate transcription in eukaryotes. • Eukaryotic cells modify mRNA after transcription. • Splicing of mRNA increases the number of different proteins an organism can produce. • Gene expression is regulated by proteins that bind to specific base sequences in DNA. • The environment of a cell and of an organism has an impact on gene expression. 7.2 Application and skills: • Application: The promoter as an example of non-coding DNA with a function. • Skill: Analysis of changes in the DNA methylation patterns.
7.3 Understandings: • Initiation of translation involves assembly of the components that carry out the process. • Synthesis of the polypeptide involves a repeated cycle of events. • Disassembly of the components follows termination of translation. • Free ribosomes synthesize proteins for use primarily within the cell. • Bound ribosomes synthesize proteins primarily for secretion or for use in lysosomes. • Translation can occur immediately after transcription in prokaryotes due to the absence of a nuclear membrane. • The sequence and number of amino acids in the polypeptide is the primary structure. • The secondary structure is the formation of alpha helices and beta pleated sheets stabilized by hydrogen bonding. • The tertiary structure is the further folding of the polypeptide stabilized by interactions between R groups. • The quaternary structure exists in proteins with more than one polypeptide chain. 7.3 Application and skills: • Application: tRNA-activating enzymes illustrate enzyme–substrate specificity and the role of phosphorylation. • Skill: Identification of polysomes in electron micrographs of prokaryotes and eukaryotes. • Skill: The use of molecular visualization software to analyse the structure of eukaryotic ribosomes and a tRNA molecule.
I. Linking the gene to the phenotype First person to suggest that genes dictate phenotypes through enzymes (proteins) was Archibald Garrod in 1909. He was a physician who hypothesized that an inherited disorder called alkaptonuria resulted from the inability to synthesize an enzyme that could break down alkapton (homogentistic acid). Reduction of phenylalanine and tyrosine. Disorders such as this were referred to as “inborn errors ofmetabolism”. Throughout the 1930’s various investigators found mutations in the organisms that they worked with including Drosophila and bread mold (Neurospora crassa) that could be attributed to defects in metabolic pathways.
One-gene: one enzyme to one gene: one protein • First general hypothesis as the result of this work. One gene: one enzyme. • Subsequent work showed that such structural proteins as Keratin, insulin were also produced by genes so the hypothesis became: One gene: one protein
3rd time’s the charm: One gene-one polypeptide • Some proteins are made of more than onepolypeptide chain, so the current hypothesis is: One gene – one polypeptide chain.
II. RNA: the bridge between genetic information and protein • RNA differs from DNA in three ways: 1.) RNA nucleotides contain the sugar ribose instead of deoxyribose. Ribose has one more hydroxyl than deoxyribose. 2.) Uracil, a pyrimidine, is unique to RNA and is similar to thymine (A, C, G, U). 3.) RNA is single stranded (much of the time; some kinds of RNA can actually fold back on itself and hydrogen bond with itself becoming double stranded: siRNA, miRNA: small interfering and micro RNA).
Two chemical languages must be connected: 4 DNA nucleotides to 20amino acids. Processes involved in going between two languages are: • Transcription: synthesis of RNA under the direction of DNA. This RNA transcript is called messenger RNA (mRNA) because it functions as a genetic messenger from DNA to ribosomes. • Translation: synthesis of a polypeptide under the direction of mRNA.
Prokaryotes vs. Eukaryotes Major difference between prokaryotes and eukaryotes due to lack of nuclei in prokaryotes. Prokaryotes: transcription and translation are coupled (ribosomes attach while transcription still occurring). Eukaryotes: transcription and translation are separated in space and time. There is time to modify mRNA: RNA processing.
III. The Genetic Code Each nucleotide cannot specify one amino acid. Two nucleotides taken together (42) can only specify 16 amino acids. But 3 nucleotides at a time can specify 64 possible code words. Soooo….genetic code has been shown to be a triplet code where each triplet of DNA nucleotides specifies an amino acid.
Definitions: • Degenerate: there is more than one basetriplet to code for one amino acid (e.g. leucine is coded for by 6 different triplets). • Universal: it is found in all living organism. Strands of DNA distinguished between sense and antisense strands. • Sense strand: coding strand and has the same base sequence as mRNA (with uracil instead of thymine). • Antisense strand: is transcribed and has the same base sequence as tRNA. (Your book calls the antisense strand the template strand because it provides the template for ordering the sequence of the bases in an RNA transcript.)
More definitions • When a DNA triplet is transcribed into mRNA, the sequence of mRNA base triplets is called a codon. If the DNA triplet is AGT, then the mRNA codon is UCA, and just to be complete, the tRNA anti-codon is AGU. • Special codons include: mRNA codon for methionine which signals the beginning of transcription = “start” codon. There are also 3 stop codons.
IV. Transcription • Major enzyme involved: RNA Polymerase II. Function: to pry the two strands of DNA apart and catalyze the addition of RNA nucleotides as nucleoside triphosphates as they base-pair along the DNA. RNA polymerases add nucleotides only to the 3’ end of the growing polymer. A transcription unit is the entire stretch of DNA that is transcribed into a single RNA molecule, from the initiation site to the termination site. In eukaryotes: a transcription unit represents a single gene. In prokaryotes: a transcription unit may include genes that code for proteins of related functions. Transcription unit is polycistronic.
B. 3 Steps in Transcription: RNA polymerase binding and initiation, elongation, and termination • Binding and Initiation: a) RNA Polymerase binding site: Promoter which includes the initiation site where mRNA synthesis begins. b) RNA Polymerase Recognition Sites: (repetitive sequences e.g. TATA box) located upstream from the promoter. c) Transcription factors: proteins (and other moleculese.g. steroid hormones) that help RNA Polymerase search for and bind to promoter regions along the DNA molecule. d) Genes, sometimes on other chromosomes, affect binding of RNA promoter region (enhancers).
Enhancers: bind proteins that are either activators or repressors
Transcription: Elongation of the RNA Strand Steps: • RNA polymerase untwists one turn of the DNA double helix at a time exposing about 10 DNA bases for pairing with RNA nucleotides. • Enzyme adds nucleotidesas RNA nucleoside triphosphates at the 3’ end of the growing RNA molecule as it continues along the double helix. (Complementary base pairing. 1st is DNA, 2nd is the mRNA: A – U; T – A; G – C; C – G)
Elongation: continued c) mRNA molecule peels away from the DNA template d) A single gene can be transcribed simultaneously by several molecules of RNA Polymerase. This allows the production of large amounts of mRNA and, therefore, proteins.
Termination of Transcription RNA Polymerase continues adding nucleotides until it reaches the termination site on the DNA. Termination site signals RNA polymerase to stop adding nucleotides and to release the RNA molecule. In prokaryotes, mRNA’s are ready for translation. In Eukaryotes, mRNA is firstmodified.
V. Post Transcriptional Modifications • Alteration of mRNA ends: a) 5’ end (end formed first): capped off with modified form of guanine nucleotide. This protects mRNA from hydrolytic enzymes. Also, in the cytoplasm, 5’ cap serves as a signal for small ribosomal subunit to attach here. b) 3’ end (synthesized last): enzyme adds poly-A-tail of 30-200 adenine nucleotides. Helps inhibit degradation of RNA.
2. RNA Splicing • Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that are never translated. These noncoding segments are interspersed between coding segments of the gene. • Noncoding sequences of DNA are called intervening sequences or introns for short. • Coding regions are called exons because they will be expressed. • Before RNA splicing, mRNA is referred to as heterogeneous nuclear RNA (hnRNA) or pre-mRNA. The pre-mRNA never leaves the nucleus.
Enzymes involved in splicing. e) Small nuclear ribonucleoproteins or snRNPs which are composed of RNA and 7 or more proteins. This RNA is called small nuclear RNA or snRNA. • An assembly of several snRNPs is called a spliceosome which cuts mRNA at specific points to release introns, and immediately joins the two exons that were adjacent to the intron. • RNA molecules that function as enzymes: Ribozymes. Some ribozymes are actually introns. Other ribozymes include parts of ribosomes and transfer RNA (tRNA). • Possible function of introns: regulatory role in the cell; allow exon rearrangements to make different proteins in different cells. Domain shuffling. Facilitates genetic recombination by increasing the chances of recombination within a gene.
Methylation • Inactive DNA tends to be highly methylated and thus is not transcribed or translated • Methyl group is a functional group with the formula CH3 • An example is with the X-chromosome • Females have two X-chromosomes but only one is active, the inactive X-chromosome is highly methylated • Once a gene is methylated it tends to stay methylated • Methylation patterns are seen in some cancers and can now be used in diagnosis and treatment
Environment has an impact on gene expression • During the Dutch Winter Hunger 1944-1945 German still controlled the Netherlands. The German blockade caused a dramatic drop in the amount of food available for the Dutch. They were consuming only about 30% of their daily caloric needs surviving off of turnips and grass. The survivors of the famine and the children that we born to survivors who were pregnant during the famine were studied • It was found that “…individuals who were prenatally exposed to famine during the Dutch Hunger Winter in 1944–45 had, 6 decades later, less DNA methylation of the imprinted IGF2 gene compared with their unexposed, same-sex siblings.” (Heijmans, et al) • IGF2 stands for insulin-like growth factor II and is important in human growth and development. This gene is maternally imprinted so if mothers were malnourished during pregnancy they passed this on to their children. • These children had an overall lower birth weight than average • This is an example of epigenetics
VI. Translation: RNA-directed synthesis of a polypeptide • The Players: a) Transfer RNA (tRNA): shuttle for specific amino acids. i) source: transcribed from DNA templates (nucleus) ii) destination: cytoplasm where translation occurs iii) structure: 1) single RNA strand that is about 80 nucleotides long. 2) It is folded back upon itself to form a molecule with a secondary structure which looks like a cloverleaf. H bonds are important. 3 loops 3) The 2-D molecule twists and folds into a compact 3-D structure that is roughly L shape. 4) One loop of 7 nucleotides includes the triplet of bases called the anticodon. The other end of the L tRNA protrudes the 3’ end which is used for the attachment site for an amino acid. Ends in CCA. 5) Anticodon: specialized base triplet that binds to a specific mRNA codon. iv) Number = 45:(should be 61 to match non-stop codons) sufficient because some tRNAs have anticodons that recognize 2 or more different codons. Relaxation of base pair rules = wobble. 6) Sections become double stranded by base pairing. 7) An extra small loop is sometimes present 8) Base paired sections are sometimes helical The above is to illustrate how tRNAs can be specific structurally for the aa that they attach.
b) tRNA Activating Enzymes: Aminoacyl-tRNA synthetase 1.Specific enzymes that match correct amino acid with the correct tRNA. • Active site fits only specific combination of amino acid and tRNA. • Catalyzes attachment of the amino acid to its tRNA in a two-step process driven by the hydrolysis of ATP. • Amino acid-tRNA complex is released and delivered to a growing polypeptide chain on a ribosome.
c) Ribosomes • Function: to facilitate the coupling of tRNA with mRNA codons during protein synthesis. • Structure: two subunits, the large and the small. Synthesized in the nucleolus. Subunits join only when they attach to mRNA molecule. • Molecular composition: 60% rRNA (most abundant RNA) and 40% protein. (By weight) • Prokaryotes vs. eukaryotes: slightly smaller in prokaryotes: 70s vs. 80s in eukaryotes where s = svedberg unit, a unit related to centrifugation density. Important in targeting antibiotics (streptomycin, tetracycline).
5. Binding sites • i) mRNA binding site • ii) P site which holds the tRNA carrying the growing polypeptide chain. • iii) A site which holds the tRNA carrying the next amino acid to be added to the chain. • iv) E site: exit site
