Transcription & Translation

1. Transcription & Translation

2. Key Concepts • After RNA polymerase binds DNA with the help of other proteins, it catalyzes the production of an RNA molecule whose base sequence is complementary to the base sequence of the DNA template strand. • Eukaryotic genes contain regions called exons and regions called introns; during RNA processing, the regions coded by introns are removed, and the ends of the RNA receive a cap and tail. • Ribosomes translate mRNAs into proteins with the help of intermediary molecules called transfer RNAs (tRNAs). • Difference between promotion of prokaryote and eukaryote protein synthesis • Each transfer RNA carries an amino acid corresponding to the tRNA’s three-base-long anticodon. • In the ribosome, the tRNA anticodon binds to a three-base-long mRNA codon, causing the amino acid carried by the transfer RNA to be added to the growing protein.

3. Overview of Transcription • A cell builds the proteins it needs from instructions encoded in its genome according to the central dogma of molecular biology. • The first step in converting genetic information into proteins is transcription, the synthesis of an mRNA version of the instructions stored in DNA. • RNA polymerase performs this synthesis by transcribing only one strand of DNA, called the template strand. • The other DNA strand is called the non-template, or coding strand, which matches the sequence of the mRNA, except that RNA has uracil (U) in place of thymine (T).

5. Characteristics of RNA Polymerase • Like the DNA polymerases, an RNA polymerase performs a template-directed synthesis in the 5′ to 3′ direction. But unlike DNA polymerases, RNA polymerases do not require a primer to begin transcription. • Bacteria have one RNA polymerase while eukaryotes have three distinct types, RNA polymerase I, II, and III.

6. Initiation: How Does Transcription Begin? • Initiation is the first phase of transcription. • However, RNA polymerase cannot initiate transcription on its own. • Sigma, a protein subunit, must first bind to the polymerase to initiate polymerization of an RNA transcript.

7. What Role Does Sigma Play in Initiation? • Sigma and RNA polymerase together form a holoenzyme, an enzyme made up of a core enzyme and other required proteins • Prokaryotic RNA polymerase is a holoenzyme made up of the core enzyme, which has the ability to synthesize RNA, and a sigma subunit. • Sigma acts as a regulatory factor, guiding RNA polymerase to specific promoter sequences on the DNA template strand.

8. Bacterial (Prokaryote) Promoters • Bacterial promoters are comprised of 4050 base pairs and have two key regions. • The –10 box is found 10 bases upstream (in the opposite direction of RNA polymerase movement during transcription) from the transcription start site (the +1 site) and consists of the sequence TATAAT. • The –35 box, consisting of the sequence TTGACA, is 35 bases upstream from the +1 site. • All bacterial promoters have a –10 box and a –35 box, the remainder of the promoter sequence varies.

10. Eukaryotic Promoters • Eukaryotes have a much more diverse and complex series of promoters than do prokaryotes. • Many of the eukaryotic promoters include a unique sequence called the TATA box, centered about 30 base pairs upstream of the transcription start site.

11. In Bacteria, Sigma Subunits Initiate Transcription • Transcription begins when sigma, as part of the holoenzyme complex, binds to the –35 and –10 boxes. • Sigma, and not RNA polymerase, makes the initial contact with DNA that starts transcription, supporting the hypothesis that sigma is a regulatory protein. • Most bacteria have several types of sigma proteins. • Each type allows RNA polymerase to bind to a different type of promoter and therefore a different kind of gene.

12. Transcription Initiation in Eukaryotes • As with bacteria, the RNA polymerase does not bind directly to the promoter. • In eukaryotes, a group of proteins called basal transcription factors bind to the DNA promoter, thus initiating transcription. • Basal transcription factors perform a similar function to bacterial sigma proteins. • However, basal transcription factors include many proteins, and they are not part of a holoenzyme.

13. What Occurs Inside the Holoenzyme? • Sigma opens the DNA double helix and the template strand is threaded through the RNA polymerase active site. • An incoming ribonucleoside triphosphate (NTP) pairs with a complementary base on the DNA template strand, and RNA polymerization begins. • Sigma dissociates from the core enzyme once the initiation phase of transcription is completed.

15. Elongation and Termination • During the elongationphase of transcription, RNA polymerase moves along the DNA template and synthesizes RNA in the 5'  3' direction. • Transcription ends with a termination phase. In this phase, RNA polymerase encounters a transcription termination signal in the DNA template. • In bacteria the transcription termination signal codes for RNA forming a hairpin structure, which causes the RNA polymerase to separate from the RNA transcript, ending transcription.

17. RNA Processing in Eukaryotes • In bacteria, the information in DNA is converted to mRNA directly. In eukaryotes, however, the product of transcription is an immature primary transcript, or pre-mRNA. Before primary transcripts can be translated, they have to be processed in a complex series of steps.

18. The Discovery of Eukaryotic Genes in Pieces • The protein-coding regions of eukaryotic genes are interrupted by noncoding regions. • To make a functional mRNA, these noncoding regions must be removed. • Exons are the coding regions of eukaryotic genes that will be part of the final mRNA product. • The intervening noncoding sequences are called introns, and are not in the final mRNA. • Eukaryotic genes are much larger than their corresponding mature mRNA.

20. RNA Splicing • The transcription of eukaryotic genes by RNA polymerase generates a primary RNA transcript that contains exons and introns. • Introns are removed by splicing. • Small nuclear ribonucleoproteins (snRNPs) form a complex called a spliceosome. This spliceosome catalyzes the splicing reaction.

22. Adding Caps and Tails to RNA Transcripts • Primary RNA transcripts are also processed by the addition of a 5′ cap and a poly(A) tail. • With the addition of cap and tail and completion of splicing, processing of the primary RNA transcript is complete. The product is a mature mRNA. • The 5' cap serves as a recognition signal for the translation machinery. • The poly(A) tail extends the life of an mRNA by protecting it from degradation.

24. An Introduction to Translation • In translation, the sequence of bases in the mRNA is converted to an amino acid sequence in a protein. • Ribosomes catalyze translation of the mRNA sequence into protein.

25. Transcription and Translation in Bacteria • In bacteria, transcription and translation can occur simultaneously. Bacterial ribosomes begin translating an mRNA before RNA polymerase has finished transcribing it. • Multiple ribosomes attached to an mRNA form a polyribosome. • In eukaryotes, transcription and translation are separated. mRNAs are synthesized and processed in the nucleus and then transported to the cytoplasm for translation by ribosomes.

27. Transcription and Translation in Eukaryotes • In eukaryotes, transcription and translation are separated. mRNAs are synthesized and processed in the nucleus and then transported to the cytoplasm for translation by ribosomes.

29. How Does an mRNA Triplet Specify an Amino Acid? • There were two hypotheses regarding the specification of amino acid sequence by a sequence of nucleotide bases: • mRNA codons and amino acids interact directly. • Francis Crick proposed that an adapter molecule holds amino acids in place while interacting directly and specifically with a codon in mRNA. • The adapter molecule was later found to be a small RNA called transfer RNA (tRNA).

31. The Characteristics of Transfer RNA • ATP is required to attach tRNA to an amino acid. • Enzymes called aminoacyl tRNA synthetases “charge” the tRNA by catalyzing the addition of amino acids to tRNAs. • For each of the 20 amino acids, there is a different aminoacyl tRNA synthetase and one or more tRNAs. • A tRNA covalently linked to its corresponding amino acid is called an aminoacyl tRNA.

33. What Happens to the Amino Acids Attached to tRNA? • Experiments with radioactive amino acids revealed that they are lost from tRNAs and incorporated into polypeptides synthesized in ribosomes. • These results inspired the use of “transfer” in tRNA’s name, because amino acids are transferred from the RNA to the growing end of a new polypeptide. The experiment also confirmed that aminoacyl tRNAs act as the interpreter in the translation process: tRNAs are Crick’s adapter molecules.

34. What Do tRNAs Look Like? • The CCA sequence at the 3' end of each tRNA is the binding site for amino acids. • The triplet on the loop at the opposite end is the anticodon that base pairs with the mRNA codon. • The secondary structure of tRNA folds over to produce an L-shaped tertiary structure. • All of the tRNAs in a cell have the same structure, shaped like an upside-down L. They vary at the anticodon and attached amino acid.

36. How Many tRNAs Are There? • There are 61 different codons but only about 40 tRNAs in most cells. • To resolve this deficit, Francis Crick proposed the wobblehypothesis. This hypothesis proposes that the anticodon of tRNAs can still bind successfully to a codon whose third position requires a nonstandard base pairing. • Thus, one tRNA is able to base pair with more than one type of codon.

37. The Structure and Function of Ribosomes • Ribosomes contain protein and ribosomal RNA (rRNA). • Ribosomes can be separated into two subunits: • The small subunit, which holds the mRNA in place during translation. • The large subunit, where peptide bonds form. • During translation, three distinct tRNAs line up within the ribosome.

38. Ribosomes and the Mechanism of Translation • All three tRNAs are bound at their anticodons to the corresponding mRNA codon. • The A site of the ribosome is the acceptor site for an aminoacyl tRNA. • The P site is where a peptide bond forms that adds an amino acid to the growing polypeptide chain. • The E site is where tRNAs no longer bound to an amino acid exit the ribosome.

40. Ribosomes and the Mechanism of Translation • The ribosome is a molecular machine that synthesizes proteins in a three-step sequence. • An aminoacyl tRNA carrying the correct anticodon for the mRNA codon enters the A site. • A peptide bond forms between the amino acid on the aminoacyl tRNA in the A site and the growing polypeptide on the tRNA in the P site. • The ribosome moves ahead three bases and all three tRNAs move down one position; the tRNA in the E site exits.

41. The Phases of Translation Translation has three phases: • Initiation • Elongation • Termination

42. Initiation • The initiation phase of translation begins at the AUG start codon. • In bacteria, the start codon is preceded by a ribosomebinding site (also called the Shine-Dalgarno sequence) that is complementary to a section of one rRNA in the small ribosomal subunit. • The interaction between the small subunit and the mRNA is mediated by initiation factors.

43. Initiation in Bacteria • Translation initiation is a three-step process in bacteria: • The mRNA binds to a small ribosomal subunit. • Theinitiator aminoacyl tRNA bearing N-formylmethionine (f-met) binds to the start codon. 3. The large ribosomal subunit binds, completing the complex. • Translation is now ready to begin.

45. Elongation • At the start of the elongation phase, the initiator tRNA is in the P site, and the E and A sites are empty. • An aminoacyl tRNA binds to the codon in the A site via complementary base pairing between anticodon and codon. • Peptide bonds form between amino acids on the tRNAs in the P and A sites. • After peptide bond formation, the polypeptide on the tRNA in the P site is transferred to the tRNA in the A site.

48. Is the Ribosome an Enzyme or a Ribozyme? • The active site of the ribosome is entirely ribosomal RNA. • Thus, ribosomal RNA catalyzes peptide bond formation and the ribosome is a ribozyme.

49. Moving Down the mRNA • Translocation occurs when elongation factors move the mRNA down the ribosome three nucleotides at a time, and the tRNA attached to the growing protein moves into the P site. • The A site is now available to accept a new aminoacyl tRNA for binding to the next codon. • The tRNA that was in the P site moves to the E site, and if the E site is occupied, that tRNA is ejected.

50. Elongation • Elongation has three steps: • Arrival of the aminoacyl tRNA. • Peptide bond formation. • Translocation.