Chapter 5: Gene Expression - Transcription

1. Chapter 5: Gene Expression - Transcription • When a protein is needed by a cell, the genetic code for that protein must be read from the DNA and processed. • A two step process: • Transcription = synthesis of a single-stranded RNA molecule using the DNA template (1 strand of DNA is transcribed). • Translation = conversion of a messenger RNA sequence into the amino acid sequence of a polypeptide (i.e., protein synthesis) • Both processes occur throughout the cell cycle. Transcription occurs in the nucleus, whereas translation occurs in the cytoplasm.

3. Transcription: How is an RNA strand synthesized? • Regulated by gene regulatory elements within each gene. • DNA unwinds next to a gene. • RNA is transcribed 5’ to 3’ from the template (3’ to 5’). • Similar to DNA synthesis, except: • NTPs instead of dNTPs (no deoxy-) • No primer • No proofreading • Adds Uracil (U) instead of thymine (T) • RNA polymerase

4. Fig. 5.2

5. Animation RNA biosynthesis

6. Three Steps to Transcription: • Initiation • Elongation • Termination • Occur in both prokaryotes and eukaryotes. • Elongation is conserved in prokaryotes and eukaryotes. • Initiation and termination proceed differently.

7. Step 1-Initiation, E. coli model: • Fig. 5.3 Each gene has three regions: • 5’ Promoter, attracts RNA polymerase -10 bp 5’-TATAAT-3’ -35 bp 5’-TTGACA-3’ • Transcribed sequence (transcript) or RNA coding sequence • 3’ Terminator, signals the stop point

8. Step 1-Initiation, E. coli model: • RNA polymerase combines with sigma factor (a polypeptide) to create RNA polymerase holoenzyme • Recognizes promoters and initiates transcription. • Sigma factor required for efficient binding and transcription. • Different sigma factors recognize different promoter sequences. • RNA polymerase holoenzyme binds promoters and untwists DNA • Binds loosely to the -35 promoter (DNA is d.s.) • Binds tightly to the -10 promoter and untwists • Different types and levels of sigma factors influence the level and dynamics of gene expression (how much and efficiency).

9. Fig. 5.4

10. Step 2-Elongation, E. coli model: After 8-9 bp of RNA synthesis occurs, sigma factor is released and recycled for other reactions. RNA polymerase completes the transcription at 30-50 bp/second. DNA untwists rapidly, and re-anneals behind the enzyme. Part of the new RNA strand is hybrid DNA-RNA, but most RNA is displaced as the helix reforms.

11. Fig. 5.4

12. Step 3-Termination, E. coli model: • Two types of terminator sequences occur in prokaryotes: • Type I (-independent) Palindromic, inverse repeat forms a hairpin loop and is believed to physically destabilize the DNA-RNA hybrid. • Type II (-dependent) • Involves  factor proteins, believed to break the hydrogen bonds between the template DNA and RNA. Fig. 5.5

13. Prokaryotes possess only one type of RNA polymerase • transcribes mRNAs, tRNAs, and rRNAs Transcription is more complicated in eukaryotes • Eukaryotes possess three RNA polymerases: • RNA polymerase I, transcribes three major rRNAs 12S, 18S, 5.8S • RNA polymerase II, transcribes mRNAs and some snRNAs • RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs *S values of rRNAs refer to molecular size, as determined by sucrose gradient centrifugation. RNAs with larger S values are larger/have a greater density.

15. Transcription of protein-coding genes by RNA polymerase II • RNA polymerase II transcribes a precursor-mRNA • We can divide eukaryotes promoter into two regions: • The core promoters elements. The best characterized are • A short sequence called Inr (Initiator) • TATA Box = TATAAAA, located at about position -30 *AT-rich DNA is easier to denature than GC-rich DNA • Promoter proximal elements (located upstream, ~-50 to -200 bp) “Cat Box” = CAAT and “GC Box” GGGCGG • Different combinations occur near different genes. • Transcription regulatory proteins (activators) and enhancers also are required.

16. Transcription regulatory proteins = Activators • High-level transcription is induced by binding of activator factors to DNA sequences called enhancers. • Enhancers are usually located upstream of the gene they control, they modulate transcription from a distance. • Can be several kb from the gene • Silencer elements and repressor factors also exist

17. Transcription of protein-coding genes by RNA polymerase II • General Transcription factors (GTFs) also are required by RNA polymerases (function is similar to sigma factor). • GTFs are proteins, assembled on the core promoter • Each GTF works with only one kind of RNA polymerase (required by all 3 RNA polymerases). • Numbered (i.e., named) to match their RNA polymerase. TFIID, TFIIB, TFIIF, TFIIE, TFIIH • Binding of GTFs and RNA polymerase occurs in a set order in protein coding genes. • Complete complex (RNA polymerase + GTFs) is called a pre-initiation complex (PIC).

18. Order of binding is: IID + IIA + IIB + RNA poly. II + IIF +IIE +IIH Fig. 5.7.

19. Production of the mRNA molecule (Fig. 5.8) Three main parts: 5’ untranslated region (5’ UTR) or leader sequence Coding sequence, specifies amino acids to be translated 3’ untranslated region ( 3’ UTR) or trailer sequence may contain information that signals the stability of the particular mRNA

20. mRNA differences between prokaryotes and eukaryotes: Prokaryotes mRNA transcript is mature, and used directly for translation without modification. Since prokaryotes lack a nucleus, mRNA also is translated on ribosomes before it is transcribed completely (i.e., transcription and translation are coupled). Prokaryote mRNAs are polycistronic, they contain amino acid coding information for more than one gene. Eukaryotes mRNA transcript is not mature (pre-mRNA); must be processed. Transcription and translation are not coupled (mRNA must first be exported to the cytoplasm before translation occurs). Eukaryote mRNAs are monocistronic, they contain amino acid sequences for just one gene.

21. Fig. 5.9. Prokaryotes and Eukaryotes

22. Production of mature mRNA in eukaryotes: • 5’ cap • After 20-30 nucleotides have been synthesized, the 5’-end of the mRNA is capped 5’ to 5’ with a guanine nucleotide (See Fig. 5.10). • Results in the addition of two methyl (CH3) groups. • Essential for the ribosome to bind to the 5’ end of the mRNA. • Poly (A) tail • 50-250 adenine nucleotides are added to 3’ end of mRNA. • Complex enzymatic reaction. • Stabilizes the mRNA, and plays an important role in transcription termination. • Introns (non-coding sequences between exons) are removed and exons (amino acid coding sequences) are spliced.

23. Animation RNA processing

24. Introns and exons: • Eukaryote pre-mRNAs often have intervening introns that must be removed during RNA processing (as do some viruses). intron = intragenic region made of non-coding DNA sequences between exons in a gene. exon = expressed DNA sequences in a gene, code for amino acids. Fig. 5.12 1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)

25. mRNA splicing of exons and removal of introns: • Introns typically begin with a 5’-GU and end with AG-3’. • Cleavage occurs first at the 5’ end of intron 1 (between 2 exons). • The now free G joins with an A at a specific branch point sequence in the middle of the intron, using a 2’ to 5’ phosphodiester bond. • Intron forms a lariat-shaped structure. • Lariat is excised, and the exons are joined to form a spliced mRNA. • Splicing is mediated by splicosomes, complexes of small nuclear RNAs (snRNAs) and proteins, that cleave the intron at the 3’ end and join the exons. • Introns are degraded by the cell.

26. Fig. 5.12

27. Fig. 5.13

28. Animation Intron Removal

29. Significance of Introns: • Introns are not junk DNA • They have important regulatory functions. • One of their primary attributes is that they enable the differential splicing of different exons, result in different protein variants with different functional properties. • Multiple proteins from a single gene. • These patterns of gene regulation are as important as the genes themselves. • They can also regulate the rate of transcription and function as a point for recombination. • They can also be lost and gained, making them a type of mobile genetic element.

30. Post-transcriptional modification, mRNA editing: Adds or deletes nucleotides from a pre-RNA, or chemically alters the bases, so the mRNA bases do not match the DNA sequence. Can results in the substitution, addition, or deletion of amino acids (relative to the DNA template). Generally cell or tissue specific. Examples occur in protozoa, slime molds, plant organelles, and mammals.

31. Genes that do not code proteins also are transcribed: • rRNA, ribosomal RNA • Catalyze protein synthesis by facilitating the binding of tRNA (and their amino acids) to mRNA. • tRNA, transfer RNA • Transport amino acids to mRNA for translation. • snRNA, small nuclear RNA • Combine with proteins to form complexes used in RNA processing (splicosomes used for intron removal).

32. 1. Synthesis of ribosomal RNA and ribosomes: • Cells contain thousands of ribosomes. • Consist of two subunits (large and small) in prokaryotes and eukaryotes, in combination with ribosomal proteins. • E. coli 70S model: • 50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins • 30S subunit = 16S (1,542 nt) + 20 proteins • Mammalian 80S model: • 60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) + 50 proteins • 40S subunit = 18S (1,900 nt) + 35 proteins • DNA regions that code for rRNA are called ribosomal DNA (rDNA). • Eukaryotes have many copies of rRNA genes tandemly repeated.

33. 1. Synthesis of ribosomal RNA and ribosomes (continued): • Transcription occurs by the same mechanism as protein-coding genes, but generally using RNA polymerase I. • rRNA synthesis requires its own array of specific transcription factors (TFs) • Coding sequences for RNA subunits within rDNA genes contain the following: • internal transcribed spacer ITS • external transcribed spacer ETS • nontranscribed spacer NTS • ITS units (analogous to introns) separate the RNA subunits through the pre-rRNA stage, whereupon ITS & ETS are cleaved out and rRNAs are assembled. • Subunits of mature ribosomes are bonded together by H-bonds. • Finally, transported to the cytoplasm to initiate protein synthesis.

34. Fig. 6.13, Mammalian example of 80S rRNA

35. Fig. 6.12

36. Fig. 5.18 2nd edition

37. 2. Synthesis of tRNA: tRNA genes also occur in repeated copies throughout the genome, and may contain introns. Each tRNA (75-90 nt in length) has a different sequence that binds a different amino acid. Many tRNAs undergo extensive post-transcription modification, especially those in the mitochondria and chloroplast. tRNAs form clover-leaf structures, with complementary base-pairing between regions to form four stems and loops. Loop #2 contains the anti-codon, which recognizes mRNA codons during translation. Same general mechanism using RNA polymerase III, promoters, unique TFs, plus post-transcriptional modification from pre-tRNA.

38. Figure 6.9

39. 3. Synthesis of snRNA (small nuclear RNA): • Form complexes with proteins used in eukaryotic RNA processing, such as splicing of mRNA after introns are removed. • Transcribed using RNA polymerase II or III. • Associated with small nuclear ribonucleoproteins (snRNPs). • Also function in regulation of transcription factors and maintenance of telomeres. U7 H/ACA