Nucleic Acids and the Encoding of Biological Information. Chapter 3

Transcription

1 Nucleic Acids and the Encoding of Biological Information Chapter 3

2 GRIFFITH S EXPERIMENT ON THE NATURE OF THE GENETIC MATERIAL In 1928, Frederick Griffith demonstrated that molecules can transfer genetic information from one organism to another.

3 AVERY, MACLEOD, AND MCCARTY S EXPERIMENT ON THE NATURE OF THE GENETIC MATERIAL Virulent bacteria (killed) DNA is extracted from heat-killed virulent cells, along with trace amounts of RNA and protein. Nonvirulent bacteria Virulent and nonvirulent bacteria The untreated extract can transform nonvirulent cells into virulent cells. Nonvirulent bacteria only Only extracts treated with the enzyme that destroys DNA were unable to transform nonvirulent bacteria.

4 Replication: Process of copying the genetic information (DNA)

5 THE CENTRAL DOGMA CARBON ATOM

6 THE CENTRAL DOGMA We learned in Chapter 1 of the central dogma DNA is transcribed into RNA and that RNA is translated into protein. The processes of transcription and translation are regulated, meaning that they can be turned on or off according to the needs of the cell function and or cell type (i.e., muscle cells). In prokaryotes, both transcription and translation occur in the cytoplasm. In eukaryotes, transcription occur in the nucleus and translation occur in the cytoplasm

7 NUCLEOTIDE STRUCTURE

8 THE FOUR BASES

9 Sugar + Base = Nucleoside Sugar + Base + Phosphate = Nucleotide

10 PHOSPHODIESTER BONDS 5 -AGCT-3

11 THE DNA DOUBLE HELIX A B

12 HYDROGEN BONDING %A = %T %G = %C

13 HYDROGEN BONDS + BASE STACKING

14 DNA REPLICATION

15 SUPERCOILING 1.DNA molecules in cells have a length far greater than the diameter of the cell itself. 2.In prokaryotic cells, the DNA is circular. 3.It forms supercoils in which the circular molecule coils upon itself, much like what happens when you twist a rubber band between your thumb and forefinger. 4. Supercoiling relieves the strain caused by topoisomerases that cleave, partially unwind, and reattach a DNA strand. 5. In doing so, supercoils also preserve the 10 base pairs per turn in the double helix.

16 6 Condensed chromatid 1400 nm in diameter Chromatin 4 Coiled chromatin fiber 300 nm in diameter 2 Nucleosome fiber 10 nm in diameter Histone proteins 1 5 Coiled coil 700 nm in diameter 3 Chromatin fiber 30 nm in diameter DNA duplex 2 nm in diameter Nucleosome

17 CHROMATIN 1.In eukaryotes, the DNA in the nucleus is linear, and each molecule forms one chromosome. 2.Again, there is a packaging problem because a chromosome is thousands of times longer than the diameter of the cell.

18 RNA WORLD HYPOTHESIS 1.Many scientists believe that RNA, not DNA, was the original information storage molecule in the earliest forms of life on Earth. 2.The reasons for thinking this are: a.rna is used in key cellular processes, including DNA replication, transcription, and translation. b.experiments have shown that RNA can evolve over time and act as a catalyst. 3.Why do cells now use DNA? Because RNA is a much less stable molecule.

19 DNA VS. RNA DNA RNA Sugar Deoxyribose Ribose Bases A, T, C, G A, U, C, G 5 end Monophosph ate Triphosphate Size Very large Smaller Strands Double Single

20 TRANSCRIPTION Template strand RNA transcript Nontemplate strand

21 TRANSCRIPTION 1.The general process of transcription is straightforward. 2.As a region of DNA unwinds, one strand is used as a template for the RNA transcript to be made. 3.The only difference is that T s are replaced with U s. 4.The enzyme responsible is RNA polymerase. 5.The new strand grows in the 5 3 direction, which means the template DNA strand is in the 3 5 direction.

22 INITIATION AND TERMINATION OF TRANSCRIPTION TATA TATA TATA TATA

23 INITIATION AND TERMINATION OF TRANSCRIPTION 1.RNA polymerase and associated proteins bind to the DNA duplex at promoter sequences. 2.Eukaryotic and archaeal promoters contain a sequence similar to TATAAA, which is known as a TATA box. 3.The first nucleotide to be transcribed is usually positioned about 25 base pairs from the TATA box. 4.RNA polymerase moves along the template strand in the 3 5 direction. 5.Transcription will continue until RNA polymerase encounters a terminator.

24 INITIATION AND TERMINATION OF TRANSCRIPTION Transcription is regulated: 1.Genes that are needed all the time in all cells (housekeeping genes) are transcribed all of the time, but 2.Most genes are transcribed only at certain times, in certain conditions, or in certain cell types.

25 Eukaryotic Promotor Recognition 1 Enhancer sequences General transcription Promoter factors bind to the promoter, and 3' transcriptional 5' activator proteins Transcriptional activator proteins bind to enhancers. 3' 5' Transcriptional start site 5' 3' General transcription factors 5' 3' DNA

26 EUKARYOTIC PROMOTOR RECOGNITION Eukaryotic Promotor Recognition 1.In bacteria, promoter recognition is mediated by a protein, sigma factor. This protein associates with RNA polymerase and facilitates its binding to specific promoters to initiate transcription. 2.In eukaryotes, transcription initiation requires the combined action of at least six proteins (general transcription factors).

27 EUKARYOTIC PROMOTOR RECOGNITION Enhancer sequence 5' 3' 2 Through looping of DNA, transcriptional activator proteins, mediator complex, RNA Pol II, and general transcription factors are brought into close proximity, allowing transcription to proceed. DNA RNA polymerase complex (Pol II) Mediator complex 5' 3' Promoter Promoter region

28 EUKARYOTIC PROMOTOR RECOGNITION Eukaryotic Promotor Recognition 1.These factors recruit RNA polymerase II (Pol II) to the site for transcription. 2.In addition, proteins bound to an enhancer sequence need to recruit a mediator complex that in turn interacts with the Pol II complex.

29 RNA POLYMERASE II ADDS NUCLEOTIDES TO THE 3 END

30 RNA Polymerase II Adds Nucleotides RNA transcript Template DNA strand RNA DNA duplex RNA polymerase complex (Pol II)

31 RNA Polymerase II Adds Nucleotides 1.Transcription takes place in what looks like a bubble that is about 14 base pairs in length. 2. The RNA-DNA duplex in the bubble is about 8 base pairs in length.

32 POLYMERIZATION REACTION

33 POLYMERIZATION REACTION The details of the polymerization reaction: 1.The incoming ribonucleotide triphosphate is recognized by the RNA polymerase and joined to the growing transcript if it base pairs correctly. In this case, the A in the DNA strand would need to be paired with a U in the transcript. 2.The RNA polymerase orients the oxygen in the hydroxyl group at the 3 end of the growing strand into a position from which it can attack the innermost phosphate of the triphosphate. 3.The 3 -OH of the growing strand attacks the high-energy phosphate of the incoming ribonucleotide, providing the energy for the reaction to take place.

34 RNA POLYMERASE IN PROKARYOTES

35 RNA POLYMERASE IN PROKARYOTES The complex in prokaryotes also forms a transcription bubble. 1.The RNA polymerase is able to separate the DNA, allow an RNA-DNA duplex to form, elongate the transcript nucleotide by nucleotide, release the finished transcript, and restore the original DNA double helix. 2.The RNA polymerase contains separate channels for the entry of the trinucleotides and DNA to be transcribed, and for the exit for the RNA transcript and transcribed DNA.

36 PRIMARY TRANSCRIPT IN PROKARYOTES

37 PRIMARY TRANSCRIPT IN PROKARYOTES 1.The RNA transcript that comes off the template DNA strand is known as the primary transcript. It contains the information of the gene that was transcribed. 2.For protein-coding genes, the primary transcript includes the information needed to direct the ribosome to produce the protein. 3.The RNA molecule that combines with the ribosome to direct protein synthesis is called mrna.

38 PRIMARY TRANSCRIPT IN PROKARYOTES 1.In prokaryotes, this relationship is simple because the primary transcript is the mrna. Both processes occur in the cytoplasm and there is no nuclear envelope to separate transcription spatially from translation. 2.It is also common in prokaryotes for the primary transcripts to contain the information for more than one gene. The mrna is then said to be polycistronic mrna.

39 PRIMARY TRANSCRIPT IN EUKARYOTES 5 Cap on Eukaryotic mrna

40 PRIMARY TRANSCRIPT IN EUKARYOTES Eukaryotic cell DNA Primary transcript (RNA) 5' cap Exon Intron Poly(A) tail The 5' end is modified by a special nucleotide called the 5' cap. mrna Spliced exons Polyadenylation adds a poly(a) tail to the 3' end. Introns are excised from the RNA strand and exons are spliced together.

41 PRIMARY TRANSCRIPT IN EUKARYOTES 1.In eukaryotes, there is a barrier between transcription and translation (the nuclear membrane). 2.The primary transcript undergoes a complex process of chemical modifications known as RNA processing. 3.Three types of chemical modification occur before the mrna is translated by the ribosome.

42 Modification 1 1. The first is the addition of a 5 cap consisting of 7- methylguanosine to the 5 end of the primary transcript. With the addition of the 5 cap, the ribosome would not recognize the mrna and translation could not occur.

43 Modification 1 7-Methylguanosine 5 Cap on Eukaryotic mrna 5'-to-5' phosphate linkage 5' end of RNA transcript The 5 cap consists of a modified base linked by its 5 carbon to the 5 end of the primary transcript by a bridge composed of three phosphates.

44 Modification 2 2. The second major modification is the addition of about 250 consecutive adenines to the 3 end of the mrna. This process is known as polyadenylation. This modification plays an important role in transcription termination as well as the export of the mrna to the cytoplasm of the cell. Both the 5 cap and the polya tail help to stabilize the RNA transcript since single-stranded nucleic acids can be unstable and susceptible to breakdown by enzymes.

45 Modification 2

46 Modification 3 3. The third modification of the primary transcript is the excision of certain sequences known as introns, leaving intact the exons. This process is known as RNA splicing. Intron removal is catalyzed by a complex of RNA and protein known as the spliceosome: About 90% of all human genes contain at least one intron

47 Modification 3

48 ALTERNATIVE SPLICING One primary transcript can code for multiple genes; which gene is formed depends on how the transcript is spliced.