Presentation on theme: "Transcription and Translation The Relationship Between Genes and Proteins."— Presentation transcript:
Transcription and Translation The Relationship Between Genes and Proteins
Table of Contents History: linking genes and proteins Getting from gene to protein: transcription Evidence for mRNA Overview of transcription RNA polymerase Stages of Transcription Promoter recognition Chain initiation Chain elongation Chain termination mRNA Synthesis/Processing References
Table of Contents (continued) Getting from gene to protein: genetic code Getting from gene to protein: translation Translation Initiation Translation Elongation Translation Termination References
History: linking genes and proteins 1900s Archibald Garrod Inborn errors of metabolism: inherited human metabolic diseases (more information)more information Genes are the inherited factors Enzymes are the biological molecules that drive metabolic reactions Enzymes are proteins Question: How do the inherited factors, the genes, control the structure and activity of enzymes (proteins)?
History: linking genes and proteins Beadle and Tatum (1941) PNAS USA 27, 499–506. Hypothesis: If genes control structure and activity of metabolic enzymes, then mutations in genes should disrupt production of required nutrients, and that disruption should be heritable. Method: Isolated ~2,000 strains from single irradiate spores (Neurospora) that grew on rich but not minimal medium. Examples: defects in B1, B6 synthesis. Conclusion: Genes govern the ability to synthesize amino acids, purines and vitamins.
History: linking genes and proteins 1950s: sickle-cell anemia Glu to Val change in hemoglobin Sequence of nucleotides in gene determines sequence of amino acids in protein Single amino acid change can alter the function of the protein Tryptophan synthase gene in E. coli Mutations resulted in single amino acid change Order of mutations in gene same as order of affected amino acids
From gene to protein: transcription Gene sequence (DNA) recopied or transcribed to RNA sequence Product of transcription is a messenger molecule that delivers the genetic instructions to the protein synthesis machinery: messenger RNA (mRNA)
Transcription: evidence for mRNA Brenner, S., Jacob, F. and Meselson, M. (1961) Nature 190, 576–81. Question: How do genes work? Does each one encode a different type of ribosome which in turn synthesizes a different protein, OR Are all ribosomes alike, receiving the genetic information to create each different protein via some kind of messenger molecule?
Transcription: evidence for mRNA E. coli cells switch from making bacterial proteins to phage proteins when infected with bacteriophage T4. Grow bacteria on medium containing heavy nitrogen ( 15 N) and carbon ( 13 C). Infect with phage T4. Immediately transfer to light medium containing radioactive uracil.
Transcription: evidence for mRNA If genes encode different ribosomes, the newly synthesized phage ribosomes will be light. If genes direct new RNA synthesis, the RNA will contain radiolabeled uracil. Results: Ribosomes from phage-infected cells were heavy, banding at the same density on a CsCl gradient as the original ribosomes. Newly synthesized RNA was associated with the heavy ribosomes. New RNA hybridized with viral ssDNA, not bacterial ssDNA.
Transcription: evidence for mRNA Conclusion Expression of phage DNA results in new phage-specific RNA molecules (mRNA) These mRNA molecules are temporarily associated with ribosomes Ribosomes do not themselves contain the genetic directions for assembling individual proteins
Transcription: overview Transcription requires: ribonucleoside 5´ triphosphates: ATP, GTP, CTP and UTP bases are adenine, guanine, cytosine and uracil sugar is ribose (not deoxyribose) DNA-dependent RNA polymerase Template (sense) DNA strand Animation of transcription
Transcription: overview Features of transcription: RNA polymerase catalyzes sugar-phosphate bond between 3´-OH of ribose and the 5´-PO 4.RNA polymerase Order of bases in DNA template strand determines order of bases in transcript. Nucleotides are added to the 3´-OH of the growing chain. RNA synthesis does not require a primer.
Transcription: overview In prokaryotes transcription and translation are coupled. Proteins are synthesized directly from the primary transcript as it is made. In eukaryotes transcription and translation are separated. Transcription occurs in the nucleus, and translation occurs in the cytoplasm on ribosomes. Figure comparing eukaryotic and prokaryotic transcription and translation.Figure
Transcription: RNA Polymerase DNA-dependent DNA template, ribonucleoside 5´ triphosphates, and Mg 2+ Synthesizes RNA in 5´ to 3´ direction E. coli RNA polymerase consists of 5 subunits Eukaryotes have three RNA polymerases RNA polymerase II is responsible for transcription of protein-coding genes and some snRNA molecules RNA polymerase II has 12 subunits Requires accessory proteins (transcription factors) Does not require a primer
Transcription: promoter recognition Transcription factors bind to promoter sequences and recruit RNA polymerase.Transcription factors bind to promoter sequences and recruit RNA polymerase DNA is bound first in a closed complex. Then, RNA polymerase denatures a 12–15 bp segment of the DNA (open complex). The site where the first base is incorporated into the transcription is numbered +1 and is called the transcription start site. Transcription factors that are required at every promoter site for RNA polymerase interaction are called basal transcription factors.
Promoter recognition: promoter sequences Promoter sequences vary considerably. RNA polymerase binds to different promoters with different strengths; binding strength relates to the level of gene expression There are some common consensus sequences for promoters:consensus sequences Example: E. coli –35 sequence (found 35 bases 5´ to the start of transcription) Example: E. coli TATA box (found 10 bases 5´ to the start of transcription)
Promoter recognition: enhancers Eukaryotic genes may also have enhancers. Enhancers can be located at great distances from the gene they regulate, either 5´ or 3´ of the transcription start, in introns or even on the noncoding strand.located One of the most common ways to identify promoters and enhancers is to use a reporter gene.
Promoter recognition: other players Many proteins can regulate gene expression by modulating the strength of interaction between the promoter and RNA polymerase. Some proteins can activate transcription (upregulate gene expression). Some proteins can inhibit transcription by blocking polymerase activity. Some proteins can act both as repressors and activators of transcription.
Transcription: chain initiation Chain initiation:Chain initiation RNA polymerase locally denatures the DNA. The first base of the new RNA strand is placed complementary to the +1 site. RNA polymerase does not require a primer. The first 8 or 9 bases of the transcript are linked. Transcription factors are released, and the polymerase leaves the promoter region. Figure of bacterial transcription initiation.bacterial transcription initiation
Transcription: chain elongation Chain elongation:Chainelongation RNA polymerase moves along the transcribed or template DNA strand. The new RNA molecule (primary transcript) forms a short RNA-DNA hybrid molecule with the DNA template.
Transcription: chain termination Most known about bacterial chain terminationbacterial chain termination Termination is signaled by a sequence that can form a hairpin loop. The polymerase and the new RNA molecule are released upon formation of the loop. Review the transcription animation.
Transcription: mRNA synthesis/processing Prokaryotes: mRNA transcribed directly from DNA template and used immediately in protein synthesis Eukaryotes: primary transcript must be processed to produce the mRNAprocessed Noncoding sequences (introns) are removed Coding sequences (exons) spliced together 5´-methylguanosine cap added 3´-polyadenosine tail added
Transcription: mRNA synthesis/processing Removal of introns and splicing of exons can occur several ways For introns within a nuclear transcript, a spliceosome is required.spliceosome Splicesomes protein and small nuclear RNA (snRNA) Specificity of splicing comes from the snRNA, some of which contain sequences complementary to the splice junctions between introns and exons Alternative splicing can produce different forms of a protein from the same gene Alternative splicing Mutations at the splice sites can cause disease Mutations Thalassemia Breast cancer (BRCA 1)ThalassemiaBreast cancer
Transcription: mRNA synthesis/processing RNA splicing inside the nucleus on particles called spliceosomes. Splicesomes are composed of proteins and small RNA molecules (100–200 bp; snRNA). Both proteins and RNA are required, but some suggesting that RNA can catalyze the splicing reaction. Self-splicing in Tetrahymena: the RNA catalyzes its own splicing Catalytic RNA: ribozymes
From gene to protein: genetic code Central Dogma Information travels from DNA to RNA to Protein Is there a one-to-one correspondence between DNA, RNA and Protein? –DNA and RNA each have four nucleotides that can form them; so yes, there is a one-to-one correspondence between DNA and RNA. –Proteins can be composed of a potential 20 amino acids; only four RNA nucleotides: no one-to-one correspondence. –How then does RNA direct the order and number of amino acids in a protein?
From gene to protein: genetic code How many bases are required for each amino acid? (4 bases) 2bases/aa = 16 amino acidsnot enough (4 bases) 3bases/aa = 64 amino acid possibilities Minimum of 3 bases/aa required What is the nature of the code? Does it have punctuation? Is it overlapping? Crick, F.H. et al. (1961) Nature 192, 1227–32. (http://profiles.nlm.nih.gov/SC/B/C/B/J/ )http://profiles.nlm.nih.gov/SC/B/C/B/J/ 3-base, nonoverlapping code that is read from a fixed point.
From gene to protein: genetic code Nirenberg and Matthaei: in vitro protein translation Found that adding rRNA prolonged cell-free protein synthesis Adding artificial RNA synthesized by polynucleotide phosphorylase (no template, UUUUUUUUU) stimulated protein synthesis more The protein that came out of this reaction was polyphenylalanine (UUU = Phe) Other artificial RNAs: AAA = Lys; CCC =Pro
From gene to protein: genetic code Nirenberg: Triplet binding assay: add triplet RNA, ribosomes, binding factors, GTP, and radiolabeled charged tRNA (figure)figure UUU trinucleotide binds to Phe-tRNA UGU trinucleotide binds to CYS-tRNA By fits and starts the triplet genetic code was worked out.triplet genetic code Each three-letter word (codon) specifies an amino acid or directions to stop translation. The code is redundant or degenerate: more than one way to encode an amino acid
From gene to protein: Translation Components required for translation: mRNA Ribosomes tRNA Aminoacyl tRNA synthetases Initiation, elongation and termination factors Animation of translation
Translation: initiation Ribosome small subunit binds to mRNA Charged tRNA anticodon forms base pairs with the mRNA codon Small subunit interacts with initiation factors and special initiator tRNA that is charged with methionine mRNA-small subunit-tRNA complex recruits the large subunit Eukaryotic and prokaryotic initiation differ slightlyEukaryoticprokaryotic
Translation: initiation The large subunit of the ribosome contains three binding sites Amino acyl (A site) Peptidyl (P site) Exit (E site) At initiation, The tRNA fMet occupies the P site A second, charged tRNA complementary to the next codon binds the A site.
Translation: elongation Elongation Ribosome translocates by three bases after peptide bond formed New charged tRNA aligns in the A site Peptide bond between amino acids in A and P sites is formedPeptide bond Ribosome translocates by three more bases The uncharged tRNA in the A site is moved to the E site.
Translation: elongation EF-Tu recruits charged tRNA to A site. Requires hydrolysis of GTP Peptidyl transferase catalyzes peptide bond formation (bond between aa and tRNA in the P site converted to peptide bond between the two amino acids) Peptide bond formation requires RNA and may be a ribozyme-catalyzed reaction
Translation: termination Termination Elongation proceeds until STOP codon reached UAA, UAG, UGA No tRNA normally exists that can form base pairing with a STOP codon; recognized by a release factor tRNA charged with last amino acid will remain at P site Release factors cleave the amino acid from the tRNA Ribosome subunits dissociate from each other Review the animation of translation