Advanced Cellular Biology Chapter 7 Notes!
Advanced Cellular Biology Chapter 7 Notes! BCMB 311
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This 6 page Class Notes was uploaded by Izabella Nill Gomez on Sunday February 7, 2016. The Class Notes belongs to BCMB 311 at University of Tennessee - Knoxville taught by Dr. Barry Bruce, Dr. J. Park in Spring 2016. Since its upload, it has received 10 views.
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Date Created: 02/07/16
Advanced Cell Bio Chapter 7 Notes! DNA does not synthesize DNA by itself, but acts like a manager, delegating tasks to workers. When a protein is needed, the nucleotide sequence is copied form an RNA segment called a gene, and the resulting RNA copies are used to make a protein. The central dogma of molecular biology is DNA to RNA to protein. Transcription/translation is a means by which cells read out or express instructions in their genes. Many identical RNA copies ca be made by the same gene, and each RNA can direct synthesis of the same protein. Successive amplification allows cells to synthesize large amounts of protein whenever necessary. Cell can regulate the expression of each of its genes according to reads. The first step to expression of genes is to copy nucleotide sequence to RNA (transcription). RNA is a linear polymer made of 4 different nucleotide subunits, linked by phosphodiester bonds. Differs from DNA--contains U instead of T, contains sugar ribose. The structure is dramatically different--DNA has 2x helix, RNA single--can fold into many shapes (like a polypeptide). Some RNAs have structural, regulatory or catalytic functions unlike DNA. Transcription begins by opening and unwinding a small portion of DNA to expose the bases on each DNA strand. 1 of 2 then acts as a template for synthesis of RNA. Ribonucleotides are added one by one to RNA chain, determined by complimentary base pairing. When a match is made, incoming ribonucleotides are covalently linked to the growing RNA by RNA polymerase. RNA chain--RNA transcript is elongated. TO leave DNA , RNA strand is displaced and DNA helix reforms-- doesn’t stay H bonded to DNA--also much shorter than DNA. RNA polymerase catalyzes the formation of phosphodiester bonds that link nucleotides together and form sugar-phosphate backbone of RNA chain. RNA polymerase moves stepwise along DNA, unwinding the helix to expose a new region of template strand for copying (5’-3’)--ATP, GTP, CTP, UTP provide energy needed for the reaction. RNA polymerase catalyzes basically the same reaction as DNA polymerase, but uses ribonucleotides. Can start replication without a primer. Likely evolved because transcription need not be as accurate as DNA replication--RNA not used for 4 permanent storage--so mistakes have minor consequences-- 10 nucleotides per 1 mistake. RNA that directs synthesis of proteins is mRNA. Usually carries information transcribed form just one gene to one protein. In bacteria, adjacent genes can be transcribed and consecutive proteins synthesized. rRNAs form structural and catalytic core of ribosomes, which translate mRNAs into protein, and tRNAs act as adaptors that select specific amino acids and hold them in place on a ribosome for incorporation into protein. miRNAs serve as key regulators of eukaryotic gene expression. Gene expression refers to the process by which information in DNA is translated into product that affects a cell/organism. When RNA polymerase collides randomly with a DNA molecule, enzyme sticks weakly to 2x helix and slides rapidly along its length. RNA polymerase latches on tightly only after it finds gene region promoter, which has specific sequence of nucleotides that is immediately upstream of starting point for RNA synthesis. Once bound tightly, RNA polymerase opens helix to expose nucleotides--one side acts as a template. Chain elongation until terminator found--transcribed onto new RNA. In bacteria, sigma factor is responsible for recognizing promoter--seen because of unique factors lying outside of the helix so no need to open helix to find it. Polarity of promoter helps determine what side of the helix RNA polymerase will use for transcription. Because RNA polymerase can only synthesize in the 5’-3’ direction on the enzyme bound, must use strand with 3’-5’ direction as template. Direction of transcription varies from gene to gene (typically one promoter/each). Eukaryotic RNA polymerases have different types-- I, II, III responsible for transcribing different types of genes. I and II for tRNA, rRNA, etc. II transcribes most genes, including those for proteins and miRNA. In eukarya, RNA polymerase needs general transcription factors which must assemble at each promoter, along with polymerase before transcription; mechanisms of transcription initiation is more elaborate--regulation can be aided by regulatory DNA sequences, DNA packed into nucleosomes. GTFs (accessory proteins) assemble on the promoter, where they position RNA polymerase and pull apart DNA double helix to expose template strand--like sigma factor. Assembly starts with TFIID to a segment of DNA helix composed mostly of T and A nucleotides (TATA box). Upon binding, TFIID causes a distortion in DNA double helix, landmark for assembly of other proteins. TATA is key in many promoters for RNA II polymerase. Usually 25 nucleotides upstream. TFIID+TATA=RNA polymerase +nTFs--transcription initiation complex--assembly order differs from one position to next. Liberation of RNA polymerase from GTFs is the addition of P groups to RNA polymerase tail--initiated from TFIIH (with protein kinase). Once transcription begins, TFs dissociate from DNA, waiting to be reused. When RNA polymerase is released after transcription, P groupsare taken from the tail by phosphatases; polymerase then ready to find a new promoter. Only dephosphorylated form of TNA polymerase can initiate transcription. Bacterial DNA is exposed in the cytoplasm with ribosomes close for protein synthesis--unlike bacteria, eukaraya must undergo RNA processing in the cytoplasm--includes capping, splicing, polyadenylation. Enzymes that are responsible ride on phosphorylated tail of RNA polymerase II and process the transcript as it emerges from polymerase. Those destined to be mRNA--undergo RNA capping which modifies the 5’ end of the transcript, the end that is synthesized first. Capped by adding atypical nucleotide Guanine with methyl group attached to 5’ end of RNA--occurs after RNA polymerase produces 25 nucleotides--before finishing. Polyadenylation provides new mRNA with special structure at the 3’ end--unlike bacteria, mRNA trimmed by enzyme that cuts RNA at a certain number of nucleotides, transcript is finished off by 2denzyme that adds repeated (A) nucleotides to cut end--poly-A tail.--Both cap and polyadenylation increases mRNA stability, mark identification and allow export to the cytoplasm. Introns are noncoding/intervening sequences--expressed/coding sequences are exons--usually shorter. RNA splicing removes intron copies from pre-mRNA and stitches exons together. Poly-A occurs after splicing/sometimes before. After splicing, mRNA is ready. Cues in introns allow for correct splicing--found near each end of an intron, same or similar in all introns. Splicing machine cuts out in “lariat” structure, formed by reaction of “A” nucleotide. Splicing carried out largely by RNA molecules--snRNAs--packaged with addition proteins to form snRNPs (small nuclear ribonucleotide proteins)--recognize splice site sequences via complimentary base- pairing between RNA components and sequences in pre-mRNA. Together, sNRPS form spliceosome--large assembly of RNA and protein molecules that carries out RNA splicing in the nucleus. Splicing can be alternative to produce distinct proteins from the same gene. TO transport only good mRNA, selection is high in the nucleus via macromolecules--“waste” degraded in nucleus and parts reused. Amount of time that mature mRNA persists in the cell affects the amount of protein it produces. Each mRNA is eventually degraded by ribonucleases (RNAses) present in the cytosol, but lifespan varies depending on nucleotide sequence of mRNA nad the type of cell. In bacteria, usually degraded rapidly (lifespan=3 minutes). In eukarya, persist longer (sometimes 10 hrs-B-globin; or 30 minutes). Lifespan contributed by 3’ untranslated region, between 3’ end of coding sequence and poly- A tail. Process of transcription universal: all cells use RNA polymerase and complimentary base-paring--RNA splicing seems to mark fundamental difference between pro/eukarya. Splicing allows for a variety of protein --but need for larger genome and must discard a large fraction of synthesized RNA without using it. Early cells might have had introns lost in prokarya during evolution--allowing for faster reproduction. Yeasts (eukarya) have few introns. Other theory is that some introns are originally parasitic mobile genetic elements that invaded early eukarya. Translation is the conversion of information in RNA to protein. Genetic code is ruled by which nucleotide sequence of a gene through mRNA is translated into the amino acid sequence of a protein. The sequence of RNA is read in groups of 2. 64 possible combinations of 3 nucleotides , but only 20 different amino acids. Code is redundant. Group of 3 nucleotides in RNA is called a codon, which specifies one amino acid. The same genetic code is used in all organisms, with slight differences in mRNA of mitochondria in fungi and protozoa. ** Mitochondria have their own DNA replication, transcription, protein-synthesis machinery (ribosomes) independent of the rest of the cell. In principle, mRNA sequences can be translated in any one of the 3 different reading frames, depending on where decoding begins. However, only 1/3 of the reading frames in mRNA specifies the correct protein. Codons in mRNA do not directly recognize the amino acids they specify, group of 3 nucleotides do not bind directly to amino acids. Adaptor molecules recognize, bind to codon at one site and amino acid at another site (tRNAs)--each about 80 nucleotides in length. RNA typically fold into 3D structures by forming base pairs between different regions--if extensive, will fold back into a 2x helix--tRNA a good example--cloverleaf structure. Undergoes further folding to form compact, L-shaped structure held by H bonds. 2 regions of unpaired nucleotides are situated at either end crucial to function of tRNAs in protein synthesis. One of these regions forms anticodon, set of 3 consecutive nucleotides that bind to complimentary codon in mRNA. Some amino acids have more than one tRNA, and some tRNAs are made so they can “wobble” at the 3 position of codon and tolerate a mismatch. The number of kinds of tRNAs differ from one species to the next. rRNA must be charged to carry out role with the correct amino acid--recognition and attachment dependent on aminoacyl-tRNA synthetases, which covalently couple each amino acid to the appropriate set of tRNA molecules. In most organisms, different synthetase/amino acid (20 in all). Each recognizes specific nucleotides in both anticodon and amino acid arm of the correct tRNA. Synthesis is a catalyzed reaction that attaches the amino acid to the 3’ end of the tRNA--is one of many reactions in cells coupled to the hydrolysis of ATP. Recognition of a codon by an anticodon on tRNA depends on the same recognition as base-pairing used in DNA replication/transcription. Ribosome--large complex made from small ribosomal proteins and rRNAs--millions in a eukaryotic cell. Eukaryotic/Prokaryotic ribosomes are similar in structure and function. 1 large/small subunit (mass is over 1 million Daltons). Small ribosomal subunit matches the tRNAs to codons of the mRNA, while the large catalyzes the formation of peptide bonds that covalently link amino acids together into a polypeptide chain. Both come together on mRNA near the 5’ end to start synthesis, then pulled like a long piece of tape. As mRNA moves forward in the 5’-3’ direction, ribosome translates. Each eukaryotic ribosome adds about 2 amino acids/sec. Bacteria even faster=20/sec. 3 binding sites for an mRNA , A, P, E site. First charged tRNA enters the A site by base pairing with the codon, amino acid then linked to the chain in the P site, exits via E. Ribosome is a ribozyme--one of the largest and most complex structures in the cell-- composed of 2/3 RNA, 1/3 protein by weight. rRNAs are responsible for overall structure and ability to choreograph/catalyze protein synthesis. rRNAs are folded into highly compact, precise 3D structures that form the core of ribosome. Ribosomal proteins fill the surface to fill gaps of folded RNA-main role is to stabilize the RNA core while permitting changes in RNA conformation. The 3 tRNA binding sites are formed primarily by rRNA--but site for catalytic peptide-bond formation made by 23s rRNA of the large subunit. Catalytic site--peptidyl transferase similar to protein enzymes--highly structures--orients elongating polypeptide and charged tRNA. Ribozymes are RNA that have catalytic activity. Initiator tRNA is a special tRNA used to start translation, carries methionine--N- terminus synthesized first (always). Usually removed later by specific protease. Loaded onto P site along with other translation initiation factors--along with initiator tRNA only one able to bind tightly to P site without the large subunit. Then, small subunit binds to 5’ end of mRNA, moves to find AUG--with tRNA bound, factors dissociate to make way for large subunit and complete assembly. In bacteria, ribosome sequences on mRNA allow for binding/start upstream of AUG. as long as binding site precedes start on mRNA, prokaryotic ribosome can bind directly to start codon in interior of mRNA--allows mRNA to be polycistronic--encode different proteins from the same mRNA. Stop codons signal the end of translation--release factor protein binds to any stop codon that reaches A site on the ribosome-- catalyzing the addition of H2O instead of amino acid to phosphorylated-tRNA--finds carboxyl end of polypeptide chain form attachment to tRNA--released, ribosome dissociates. **Most proteins require chaperone proteins to fold correctly in the cell. Synthesis of most protein molecules takes between 20 seconds and several minutes. Multiple ribosomes bind usually to each mRNA being translated. Multiple ribosomes can work simultaneously on a single mRNA to make more proteins--operate both in bacteria and eukarya, but bacteria can speed up faster--no processing--can start synthesis before the end of transcription. Many most effective antibiotics inhibit bacterial RNA and protein synthesis. Many common antibiotics first isolated from fungi. Cells have enzymatically specialized pathways that break down proteins into amino acids (proteolysis). Proteases act by hydrolyzing peptide bonds between amino acids. In eukarya, proteasomes break down proteins, present in cytosol and nucleus--contains central cylinder of proteases whose active sites face the inner chamber. Each end of the cylinder is stoppered by a large protein complex that binds proteins destined for degradation and unfold them down to the inner chamber by using ATP. Ubiquitin-tagged proteins are marked for destruction. Many steps between DNA and protein. Final concentration of protein in a cell depends on rate at which each of many steps is carried out. Many proteins also need post-translational modifications that included covalent modifications (such as phosphorylation), binding of small molecular cofactors or association with other subunits--usually needed to become fully functional. RNA world existed before cells with DNA and proteins appeared--RNA--serves largely as intermediate between genes and proteins--both store genetic information and catalyzed chemical reactions. Origin of life requires that molecules possess ability to catalyze reactions that lead directly/indirectly to production of more molecules. Life requires autocatalysts. Most versatile catalysts today are proteins--but cannot reproduce themselves. RNA can act as a catalyst with unique folded shapes--not many , but those that are catalytic serve a fundamental role in gene expression-- especially during translation. RNA is thought to predate DNA in evolution. Evidence can be seen within chemical properties (ribose-deoxyribose)--deoxyribose is harder to make--ribose is composed of simple carbs and glucose. DNA later served as a better permanent repository for genetic information. 2x helix makes easier to repair/more stable than RNA--good suggestion as to why RNA replaced.
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