Transcription and Translation

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 • 1900’s Archibald Garrod • Inborn errors of metabolism: inherited human metabolic diseases (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) Nature190, 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 (15N) and carbon (13C). • 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´-PO4. • 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.

Transcription: RNA Polymerase • DNA-dependent • DNA template, ribonucleoside 5´ triphosphates, and Mg2+ • 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

Stages of Transcription • Promoter Recognition • Chain Initiation • Chain Elongation • Chain Termination

Transcription: promoter recognition • 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: • 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. • 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: • 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.

Transcription: 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 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 mRNA • 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. • 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 • Mutations at the splice sites can cause disease • Thalassemia • Breast cancer (BRCA 1)

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 acids—not 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) Nature192, 1227–32. (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) • UUU trinucleotide binds to Phe-tRNA • UGU trinucleotide binds to CYS-tRNA • By fits and starts the triplet genetic code was worked out. • 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 slightly

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 tRNAfMet 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 formed • 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

Transcription and Translation