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Genes: Structure Replication and Expression

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1 Genes: Structure Replication and Expression
Chapter 12 Genes: Structure Replication and Expression

2 Replication: During mitotic division information is duplicated by DNA replication and is passed on to next generation daughter cells has exavcyt replica of the parent DNA

3 Role of DNA in Protein synthesis
DNA and protein synthesis involves: Transcription- yields a ribonucleic acid (RNA) copy of specific genes Translation- uses information in messenger RNA (mRNA) to synthesize a polypeptide. Protein synthesis is assisted by RNA (tRNA) and ribosomal RNA (rRNA)

4 Nucleic Acids

5 Nucleic Acid Structure Deoxyribonucleic Acid (DNA)
polymer of nucleotides contains the bases adenine, guanine, cytosine and thymine sugar is deoxyribose molecule is usually double stranded

6 DNA is a double-stranded molecule twisted into a helix (think of a spiral staircase).
Each spiraling strand, comprised of a sugar-phosphate backbone and attached bases, is connected to a complementary strand by non-covalent hydrogen bonding between paired bases. The bases are adenine (A), thymine (T), cytosine (C) and guanine (G). A and T are connected by two hydrogen bonds. G and C are connected by three hydrogen bonds.

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9 DNA Structure – Two Complementary Strands
base pairing Adenine (purine) and thymine (pyrimidine) pair by 2 hydrogen bonds Guanine (purine) and cytosine (pyrimidine) pair by 3 hydrogen bonds major and minor grooves form when the 2 strands twist around each other

10 Nucleic Acid Structure Ribonucleic Acid (RNA)
polymer of nucleotides contains the bases adenine, guanine, cytosine and uracil sugar is ribose most RNA molecules are single stranded

11 RNA Structure three different types which differ from each other in function and in structure messenger RNA (mRNA) ribosomal RNA (rRNA) transfer RNA(tRNA)

12 The Organization of DNA in Cells
In most bacteria DNA is a circular, double helix further twisting results in supercoiled DNA in bacteria the DNA is associated with basic proteins help organize the DNA into a coiled chromatin like structure

13 DNA Replication

14 DNA Replication complex process involving numerous proteins which help ensure accuracy the 2 strands separate, each serving as a template for synthesis of a complementary strand synthesis is semi-conservative; each daughter cell obtains one old and one new strand

15 DNA Replication bidirectional from a single origin of replication

16 DNA replication (arrows) occurs in both directions from the origin of replication in the circular DNA found in most bacteria.

17 Rolling Circle Replication
some small circular genomes (e.g., viruses and plasmids) replicated by rolling-circle replication Animation illustrating DNA replication by complementary base pairing A single-stranded tail, often composed of more than one genome copy, is generated and can be converted to the double-stranded form by synthesis of a complementary strand. The “free end” of the rolling circle strand is probably bound to the primosome.

18 Genes

19 Gene Structure Gene reading frame
the basic unit of genetic information also defined as the nucleic acid sequence that codes for a polypeptide, tRNA or rRNA linear sequence of nucleotides codons are found in mRNA and code for single amino acids reading frame organization of codons such that they can be read to give rise to a gene product

20 Importance of Reading Frame
Figure 12.16

21 Genes that Code for Proteins
template strand directs RNA synthesis promoter is located at the start of the gene is the recognition/binding site for RNA polymerase functions to orient polymerase leader sequence is transcribed into mRNA but is not translated into amino acids Shine-Delgarno sequence important for initiation of translation

22 Genes that Code for Proteins
The Coding Region: begins with the DNA sequence from 3´-TAC-5´ produces codon AUG which codes for N-formylmethionine, a modified amino acid used to initiate protein synthesis in bacteria ( check fig.) coding region ends with a stop codon, immediately followed by the trailer sequence which contains a terminator sequence used to stop transcription

23 Bacterial Gene Structure

24 Genes That Code for tRNA and rRNA
tRNA/rRNA genes have promoter (recognition/binding site for RNA polymerase), leader (is transcribed into mRNA), coding region, spacer and trailer regions (contains a terminator sequence used to stop transcription) during maturation process. leader, spacer, and trailer removed during maturation process Figure 12.19a:

25 rRNA genes have promoter, leader, coding, spacer, and trailer regions
spacer and trailer regions may encode tRNA molecules Figure 12.19b:

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27 Transcription

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29 Transcription RNA is synthesized under the direction of DNA
RNA produced has complementary sequence to the template DNA three types of RNA are produced mRNA carries the message for protein synthesis tRNA carries amino acids during protein synthesis rRNA molecules are components of ribosomes

30 Transcription in Bacteria…
Definitions to understand protein synthesis: in most bacterial RNA polymerases: Holoenzyme can begin transcription> What is Holoenzyme??* the core enzyme is composed of 5 chains and catalyzes RNA synthesis the sigma factor has no catalytic activity but helps the core enzyme recognize the start of genes *holoenzyme = core enzyme + sigma factor only the holoenzyme can begin transcription

31 Transcription in Bacteria….
Transcription in Bacteria is catalyzed by a single RNA polymerase. a reaction similar to that catalyzed by DNA polymerase for DNA syntehsis. ATP,GTP,CTP and UTP are used to produce a complementary RNA copy of the template DNA sequence

32 http://www. vidoemo. com/yvideo. php

33 Transcription Process

34 Transcription Initiation
Promoter site where RNA polymerase binds to initiate transcription & is not transcribed

35 Transcription Elongation
after binding, RNA polymerase unwinds the DNA transcription bubble produced moves with the polymerase as it transcribes mRNA from template strand within the bubble a temporary RNA:DNA hybrid is formed

36 Coupled Transcription and Translation in Prokaryotes

37 Proteins

38 The Genetic Code mRNA sequence is translated into amino acid sequence of polypeptide chain (process = translation). an understanding of the genetic code is necessary before translation is studied.

39 Organization of the Code
code degeneracy up to six different codons can code for a single amino acid sense codons the 61 codons that specify amino acids stop (nonsense) codons the three codons used as translation termination signals do not encode amino acids

40 Translation

41 Translation Translation of mRNA into protein:
synthesis of polypeptide is directed by sequence of nucleotides in mRNA Ribosome: 70S ribosomes = 30S + 50S subunit site of translation polyribosome (polysome) – complex of mRNA with several ribosomes

42 Translation of mRNA into protein:
Three phases: Initiation Elongation Termination

43 During translation, the mRNA is "read" according to the genetic code which relates the DNA sequence to the amino acid sequence in proteins Each group of three base pairs in mRNA constitutes a codon, and each codon specifies a particular amino acid (hence, it is a triplet code). The mRNA sequence is thus used as a template to assemble—in order—the chain of amino acids that form a protein.

44 Transfer RNA (tRNA) and Amino Acid Activation
The tRNA molecules are adaptor molecules—they have one end that can read the triplet code in the mRNA through complementary base-pairing, and another end that attaches to a specific amino acid attachment of amino acid to tRNA is catalyzed by aminoacyl-tRNA synthetases

45 The translation of mRNA begins with the formation of a complex on the mRNA (Fig. below).
First, three initiation factor proteins (known as IF1, IF2, and IF3) bind to the small subunit of the ribosome. This preinitiation complex and a methionine-carrying tRNA then bind to the mRNA, near the AUG start codon, forming the initiation complex.

46 The Ribosome

47 Methionine (Met) is the first amino acid incorporated into any new protein, however, it is not always the first amino acid in translation of protein. In many proteins, methionine is removed after translation.

48 The large ribosomal subunit binds to this complex, which causes the release of IFs (initiation factors) once the initiation complex is formed on the mRNA The large subunit of the ribosome has three sites at which tRNA molecules can bind: The A (amino acid) site is the location at which the aminoacyl-tRNA anticodon base pairs up with the mRNA codon, ensuring that correct amino acid is added to the growing polypeptide chain.

49 The P (polypeptide) site is the location at which the amino acid is transferred from its tRNA to the growing polypeptide chain. Finally, the E (exit) site is the location at which the "empty" tRNA sits before being released back into the cytoplasm to bind another amino acid and repeat the process.

50 The initiator methionine tRNA is the only aminoacyl-tRNA that can bind in the P site of the ribosome, and the A site is aligned with the second mRNA codon. The ribosome is thus ready to bind the second aminoacyl-tRNA at the A site, which will be joined to the initiator methionine by the first peptide bond.

51 Elongation of the Polypeptide Chain
The next phase in translation is known as the elongation phase . Elongation cycle is the sequential addition of amino acids to growing polypeptide & consists of three phases aminoacyl-tRNA binding transpeptidation reaction Translocation The above process need several Elongation factors ( EF)

52 ………Elongation First, the ribosome moves along the mRNA in the 5'-to-3'direction, which requires the elongation factor G, in a process called translocation

53 …..Elongation Cycle The tRNA which corresponds to the second codon can then bind to the A site, a step that requires elongation factors (in E. coli, these are called EF-Tu and EF-Ts) and GTP (guanosine triphosphate ) as an energy source for this acitivity. Upon binding of the tRNA-amino acid complex in the A site, GTP is cleaved to form guanosine diphosphate (GDP), then released along with EF-Tu to be recycled by EF-Ts for the next round. elongation cycle of protein synthesis

54 ……….Elongation In the next step, peptide bonds between the now-adjacent first and second amino acids are formed through a peptidyl transferase activity. After the peptide bond is formed, the ribosome shifts, or translocates, again, thus causing the tRNA to occupy the E site. The tRNA is then released to the cytoplasm to pick up another amino acid. The A site is now empty and ready to receive the tRNA for the next codon.

55 ….Elongation This process is repeated until all the codons in the mRNA have been read by tRNA molecules & the amino acids attached to the tRNAs have been linked together in the growing polypeptide chain in the appropriate order. At this point, translation must be terminated, and the nascent protein must be released from the mRNA and ribosome.

56 Final Phase in Elongation Cycle − Translocation
Three simultaneous events: peptidyl-tRNA moves from A site to P site ribosome moves down one codon empty tRNA leaves P site

57 Termination of Translation/protein synthesis:
Three termination codons ( Non-sense or stop codon) that are employed at the end of a protein-coding sequence in mRNA: UAA, UAG, and UGA No tRNAs recognize these codons. Instead, release factors (RFs) helps in recognition of stop codons. Release factors are protein which binds and facilitates release of the mRNA from the ribosome and subsequent dissociation of the ribosome.

58 Several ribosome can align on one mRNA strand and forms several polypeptide chains each with 20 or more amino acids.

59 Prokaryotic and Eukaryotic Translation
The translation process is very similar in prokaryotes and eukaryotes. Although different elongation, initiation, and termination factors are used, the genetic code is generally identical. In bacteria, transcription and translation take place simultaneously, and mRNAs are relatively short-lived.

60 In eukaryotes, mRNAs have highly variable half-lives,
are subject to modifications, and must exit the nucleus to be translated.

61 References


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