Transcription and Translation

1. Transcription and Translation Genes

2. The structure of a prokaryote gene

3. Gene • Consensus sequences • - 10 and -25 upstream from the start • 3’ TAC – start or initiator codon • Establishes the reading frame – ORF – open reading frame • Bases grouped in 3’s triplet code • Proceeds to stop ( ATT , ATC, ACT or termination sequence in prokaryotes

4. The Pribnow Box and Shane -Dalgarno • The RNA binding site has a consensus sequence of 5’ TATAAT 3’ ( -) and 3’ ATATTA 5’ (+) • This is where the DNA begins to become unwound for transcription • The initially transcribed sequence of the gene may not reflect doing but may be a leader sequence. • The prokaryotes usually contain a consensus sequence known as the Shane Delgarno which is complememtary to the 16s rRNA on the ribosome ( small subunit ) • The leader sequence also may regulate transcription

5. Promoters are at the beginning of the Gene • RNA polymerase recognizes a binding site in front of the gene. This is referred to as upstream of the gene. • The direction of transcription is referred to as downstream • Different genes have different promoters. IN E. coli the promoters have two functions • The RNA recognition site for transcription which is the consensus sequence for prokaryotes is 5’ TTGACA3’ ( Watson strand) which means on the reading strand 3’ AACTGT5’ ( Crick strand)

6. Genes and Gene Expression • Genes are written in a code consisting of groups of three letters called triplets. • There are four letters in the DNA alphabet. There are 64 possible arrangements of the four letters in groups of three • The triplets specify amino acids for the synthesis of proteins from the information contained in the gene

7. Genes - Continued • Genes can also specify t- RNA or r- RNAs • The gene begins with a start triplet and ends with a stop. The bases between the start and the stop are called an open reading frame, ORF. • The information in the gene is transcribed by RNA polymerase. • It reads the gene from 3’ to 5’ • The template strand is now referred to as the CRICK strand and the nontemplate strand is now known as the WATSON strand • DNA sequences are stored in data bases as the WATSON strand Reference - COLD SPRING HARBOR - 2003

8. Prokaryote Genes are • Continuous • They do not contain introns like eukaryote genes • The gene consists of codons that will determine the sequence of amino acids in the protein • At the end of the gene there is a terminator sequence rather than an actual stop • The terminator may be at the end of a trailer sequence located downstream from the actual coding region of the gene

9. Transcription Begins • DNA is read 3’ to 5’ and m RNA is synthesized 5’ to 3’ • 3’ TAC is the start triplet • This produces a complementary mRNA message 5’ AUG 3’ – • Groups of three bases in the messenger RNA formed are referred to as CODONS

10. RNA POLYMERASE

11. RNA Polymerase • There are approximately 2000 molecules of RNA Polymerase per bacterial cell • E. coli RNA polymerase is composed of six subunits with a molecular weight of over 400,000 Da

12. RNA POLYMERASE • Enzyme has been described as a claw • The Beta portions of the claw form the pincers • The Alpha portion are on the other end of the claw • The Omega portion is wrapped around the Beta portion

13. RNA Polymerase Action • Does not require a primer in comparison to DNA Polymerase • Binds to the promoter sequence and opens the double strands of DNA • Builds a chain of RNA by connecting the 5’ end of a nucleotide to the 3’ end of the nucleotide in front of it

14. Language • RNA polymerase utilizes the sequence of the template strand in DNA language to build a complementary copy of RNA • The strand that is the template is referred to as the transcribed strand • The strand with the same sequence as RNA with the exception of U instead of T is referred to as the coding strand

15. RNA Polymerase Structure – Core enzyme

16. Polymerase sub units • α2: the two α subunits assemble the enzyme and recognize regulatory factors. • These compose the core enzyme • β: this has the polymerase activity (catalyzes the synthesis of RNA) which includes chain initiation and elongation. • β': binds to DNA (nonspecifically). • ω: restores denatured RNA polymerase to its functional form in vitro. It has been observed to offer a protective/chaperone function to the β' subunit

17. RNA Polymerase Holoenzyme

18. Sigma Factors • A sigma factor (σ factor) are proteins that are required for the binding of the RNA polymerase to the promoter so that transcription can be initiated. They are also active in the elongation process.

20. Transcription and the Initiation Complex • The core enzyme binds to the holoenzyme • This is called promoter recognition • The sigma factors first bins to the -10 region • When the Beta pincer closes around the DNAto form the active site channel around the DNA, this opens the strands

21. Rifampin • The initiation somplex can be blocked by the antibiotic Rifampin • Rifampin binds to the RNA Polymerase at the active site channel is such a way that it blocks the elongation of the RNA

22. Mutations, Antibiotics, and Antibiotic Resistance

23. Initiation, Elongation, Termination

24. Transcriptional overview • http://vcell.ndsu.nodak.edu/~christjo/vcell/animationSite/transcription/elongation.htm

25. Rho independent( Factor dependent) • The termination site consists of two sequences • The first is an inverted repeat • When the inverted repeat is transcribed it forms a hairpin • The inverted repeat is followed by a string of AAAAAAAAAAAs • The RNA molecule will have terminal UUUUs • AU pairs are less stable and this causes the release of the RNA molecule from the DNA

26. Termination- Factor Independent

27. Factor Dependent • Rho Dependent ( Rho the most universal factor in prokaryote cells). There are other factors • Rho functions in the synthesis of RNAs that are not being translated • Rho forms a ring that binds to a sequence in the RNA called the rut site • Binds to the RNA and affects the action of RNA polymerase

28. Eukaryote Transcription and Transcription Factors

29. Genes for t RNAs and r RNAs • The genes for t RNAs have a promoter and transcribed leader and trailer sequence that are removed prior to their utilization in translation. Genes coding for tRNA may code for more than a single tRNA molecule • The segments coding for r RNAs are separated by spacer sequences that are removed after transcription.

31. t-RNA • The acceptor stem includes the 5' and 3' ends of the tRNA. • The 5' end is generated by RNase P • The 3' end is the site which is charged with amino acids for translation. • Aminoacyl tRNA synthetases interact with both the acceptor 3' end and the anticodon when charging tRNAs. • The anticodon matches the codon on mRNA and is read 3’ to 5’

32. t- RNA • Found in the cytoplasm • Amino acyl t- RNA synthetase is an enzyme that enables the amino acid to attach to t-RNA • Also activates the t- RNA • Clover leaf has a stem for attachment to the amino acid and an anticodon on the bottom of the clover leaf

33. t- RNA Common Features • a CCA trinucleotide at the 3' end, unpaired • four base-paired stems, and • One loop containing a T-pseudoU-C sequence and another containing dihydroU.

34. tRNA • tRNAs attach to a specific amino acid and carry it to the ribosome • There are 20 amino acids • 61 different codons for these amino acids and 61 tRNAs • The anticodon is complementary to the codon • Binds to the codon with hydrogen bonds

35. Ribosomal genes • Very similar to the structure of protein genes

36. tRNA and rRNA genes • The genes for rRNA are also similar to the organization of genes coding for proteins • All rRNA genes are transcribed as a large precursor molecule that is edited by ribonucleases after transcription to yield the final r RNA products

37. Ribosomal RNA • Combines with specific proteins to form ribosomes • Serves as a site for protein synthesis • Associated enzymes and factors control the process of translation

38. Prokaryote Ribosomes • Ribosomes are small, but complex structures, roughly 20 to 30 nm in diameter, consisting of two unequally sized subunits, referred to as large and small which fit closely together as seen below. • A subunit is composed of a complex between RNA molecules and proteins; each subunit contains at least one ribosomal RNA (rRNA) subunit and a large quantity of ribosomal proteins. • The subunits together contain up to 82 specific proteins assembled in a precise sequence.

39. 30s and 50s Ribosomal subunits

40. Prokaryote ribosomal RNA

41. Prokaryote ribosomes – polysomes- the process of translation

42. Prokaryote transcriptionand translation • Prokaryote transcription and translation take place in the cytoplasm • All necessary enzymes and molecules are present for the transcription and translation to take place

43. Translation • A molecule of messenger RNA binds to the 30S ribosome ( small ribosomal unit) at the Shine Dalgarno sequence • This insures the correct orientation for the molecule • The large ribosomal sub unit locks on top

44. T RNA and Amino acyl t RNA synthetase( Preparation of tRNAs) • The enzyme amino acyl t RNA Synthetase connects the amino acid to its specific t RNA to form cognate t RNA • Hydrolyzes ATP to transfer the amino acid to the 3’OH end of the t RNA • The acceptor arm of the t RNA ends in the sequence CCA

45. The Ribosome • There are four significant positions on the ribosome • EPAT • When the 5’ AUG 3’ of the mRNA is on the P site the t-RNA with the anticodon, 5’UAG3’ forms a temporary bond to begin translation

46. Ribosomal Sites • T site – the 5’ end of the messenger RNA enters the ribosome • A site – acceptor site -the relationship of the mRNA to the ribosome is stabilized at this site – the amino acylated t RNA is bound to the mRNA • P site the peptide bonds are formed between the amino acids on the t RNAs • E site - The mRNA moves to the final position on the ribosome as the tRNA is released

47. EPAT

48. Ribosomal structure

50. Translation Initiation Region • Should contain the initiator codon( start) AUG, may also be GUG • This may be at the start of the gene or in an Untranslated leader sequence upstream of Gene ( UTR) • Shine Delgarno named for two scientists who discovered it at -35 binds to the 30s ribosomal subuit ( 16srRNA)