5 dna rna protein synthesis
TRANSCRIPT
DNA, RNA, and Protein Synthesis
Importance and Structure of DNA: Deoxyribo-Nucleic Acid
Historical Review: 1900’s – Morgan’s studies with fruit flies
showed that genes were located on chromosomes and chromosomes consisted of protein and DNA
1952- Hershey-Chase demonstrated that DNA (not protein) was the genetic material of a viral phage
Figure 16.2a The Hershey-Chase experiment: phages
Figure 16.2b The Hershey-Chase experiment
Phages Infecting a bacterium
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Figure 16.1 Transformation of bacteria – Griffith (and later Avery, McCarty and MacLeod)
The Structure of DNA Nucleotide monomers:
Phosphate Pentose Sugar (C5) –
Deoxyribose Sugar Organic Nitrogen Base :
• Cytosine (C)• Adenine (A)• Guanine (G)• Thymine (T)
Structure of DNA cont’ Polynucleotide chain
with linkage via phosphates to next sugar, with nitrogen base away from backbone of
Phos-sugar-phos-sugar
Dehydration synthesis
Beginning of the 1950’s several labs Beginning of the 1950’s several labs were studying the structure of DNAwere studying the structure of DNA
Maurice Wilkins & Rosalind Franklin X-ray crystallography: x-rays pass
through pure DNA and diffraction of x-rays were then examined on film
James Watson and Francis Crick did not have the expertise of Franklin and were without proper photos until………
Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA
Watson and CrickFigure 16.0 Watson and Crick Figure 16.0x James Watson
April 1953 – Classical one page paper in Nature by Watson and Crick
A double helix – 2 polynucleotide strands Sugar-phosphate chains of each strand
are like the side ropes of a rope ladder Pairs of nitrogen bases, one from each
strand, form the rungs or steps The ladder forms a twist every 10 bases
(all from x-ray studies!)
Figure 16.5 The double helix
Internal Structure of DNA: Purine and pyridimine? REMEMBER X-RAY DATA
Confirms Erwin Chargaff’s Rules Confirms Erwin Chargaff’s Rules
# of Adenine = to # of thymine
# of guanine equal to # of cytosine
This dictates the combinations of N-bases that form steps/rungs
Does not restrict the sequence of bases along each DNA strand
Information storage in DNA The 4 nitrogenous bases are the
“alphabet” or code for all the traits the organism possesses
Different genes or traits vary the sequence and length of the bases
ATTTCGGAC vs. GGGATTCTAG vs. GATC
Replication/Duplication of DNA Due to complimentary base paring – one
strand of DNA determines the sequence of the other strand
Therefore, each strand of double stranded DNA acts as a template
The double helix first unwinds – controlled by enzymes –and uses new nucleotides that are free in the nucleus to copy a complimentary strand off the original DNA strand
Figure 16.7 A model for DNA replication: the basic concept (Layer 1)
Figure 16.7 A model for DNA replication: the basic concept (Layer 2)
Figure 16.7 A model for DNA replication: the basic concept (Layer 3)
Figure 16.7 A model for DNA replication: the basic concept (Layer 4)
Figure 16.8 Three alternative models of DNA replication
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 1)
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 2)
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 3)
Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)
There are a series of enzymes that control DNA replication – enzymes which:
Uncoil the original double helix strand via a helicase
Single-strand binding protein keeps helix apart so replication can start
Prime an area to start replication – primase except it adds RNA nucleotides at first
Polymerase to join individual nucleotides (dehydration synthesis)
Ligases to join short segments
DNA REPLICATION
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Figure 16.10 Origins of replication in eukaryotes
Figure 16.11 Incorporation of a nucleotide into a DNA strand
The carbons of the deoxyribose sugar are numbered
#3 carbon attached to an -OH group
#5 carbon holds the phosphate molecule of that nucleotide
#3 ready to bond with another nucleotide to form a polynucleotide link (5’ to3’)
Notice complimentary strand in opposite direction (5’ to 3’)
DNA always grows 5’ to 3’ never 3’ to 5’
Antiparallel Arrangement of Double Strands
Definitions Origins of Replication – where
replication of the DNA molecule begins Bacteria – circular DNA – 1 origin of
replication (RF) Eukaryotes – multiple origins of
replication (ORFS)• ORF = Replication Fork
More Definitions DNA Polymerases – enzymes that catalyze
DNA replication Leading Strand – Synthesized
continuously towards the replication fork by the DNA polymerase in one long fashion
Lagging Strand – Synthesized by short fragments away from the replication fork by the DNA polymerase
Definitions Cont’ Ligase – combines (joins) short fragments Primer – starts replication of DNA (in this
case it’s RNA) Primase – an enzyme that joins the RNA
nucleotides to make the primer Helicase – an enzyme that untwists the
double helix at the replication fork Nuclease – a DNA cutting enzyme
DNA REPLICATION -VIDEO
16-10-DNAReplication.swf
Figure 16.13 Synthesis of leading and lagging strands during DNA replication
Figure 16.14 Priming DNA synthesis with RNA
Figure 16.15 The main proteins of DNA replication and their functions
Figure 16.16 A summary of DNA replication
Figure 16.17 Nucleotide excision repair of DNA damage
A PROBLEM!
The end of the leading strand was initiated with an RNA primer Normally removed by
other DNA polymerase Removal of gaps by
DNA Polymerase doesn’t work on lagging strand end RNA primer removed
with no replacement A GAP! SHORTER AND SHORTER
FRAGMENTS?
Prokaryotes have circular DNA – no problem at ends (there aren’t ANY! Eukaryotes – have special terminal sequences of 6
nucleotides that repeat from 100-1000 times with no genes included Telomers
Protect more internal gene materials from being eroded
Germ cells / sex cells have a special enzyme (telomerase) that actually restore shortened Telomers
Somatic cells – telomer continues to shorten and may play a role in aged cell death
Cancer cells A telomerase prevents very short lengths
Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes
Ribonucleic Acid (RNA) Structure of RNA
Nucleotide monomer• Phosphate• Pentose sugar = ribose (extra oxygen)• Nitrogenous base (A/G/C/U)• Single stranded• 3 types (mRNA, tRNA, rRNA)
Synthesis of RNA - transcription DNA acts as a template, but only one strand of
DNA utilized at a given time This exposed strand is controlled by specific
enzymes that pair the DNA nucleotides with free RNA nucleotides which are also present in the nucleus
These RNA nucleotides form a single stranded RNA nucleic acid
DNA = ATTGGCT RNA = UAACCGA Short segments of DNA are transcribed at a time
with start and stop messages
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 1)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 2)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 3)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 4)
Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 5)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 1)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 2)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 3)
Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 4)
Figure 17.6 The stages of transcription: elongation
Three types of RNA mRNA : messenger RNA
Transcribed from a specific segment of DNA which represents a specific gene or genetic unit
tRNA : transfer RNA Transcribed from different segments of DNA and
their function is to find a specific amino acid in the cytoplasm and bring it to the mRNA
rRNA : ribosomal RNA Transcribed at the nucleolus - with proteins
function as the site of protein synthesis
Three types of RNA
Protein Synthesis = Translation Ribosomes = sites of protein synthesis
30% - 40% protein 60% - 70% RNA (rRNA) Assembled in nucleus and exported via nuclear
pores Antibiotics can paralyze bacterial ribosomes, but
not eukaryotic ribosomes (not targeting them) 2 ribosomal subunits –a large and a small Small subunit has been used as a means of
classifying different bacteria and different invertebrates (16S)• Eukaryotes – 18S
There are three sites on the ribosome that are involved in protein synthesis
Ribosomes bring mRNA together with amino acid bearing tRNA’s
Three ribosomal sites P Site (peptidyl-tRNA) holds the tRNA carrying
the growing peptide chain after several amino acids have been added
A site (aminoacyl-tRNA) holds the next single amino acid to be added to the chain
E site (exit site) site where discharged tRNA minus amino acids leave ribosome
Figure 17.15 Translation – the basic concept
Preparation of Eukaryotic mRNA
RNA splicing- a cut and paste job to remove nucleotides from transcribed mRNA 8000 nucleotides transcribed but the
average gene contains 1200+ nucleotides
Long non-coding segments (introns) interspersed between coding segments (exons) expressed via amino acids
Figure 17.17 The initiation of translation
Figure 17.18 The elongation cycle of translation
Protein Synthesis (cont’)Initiation – elongation - termination
Starting at one end of the mRNA, the small ribosomal subunit associates with the mRNA and accepts the first tRNA with its activated amino acid attached = Initiation
tRNA associate with a triplet codon exposed on the mRNA – these are 3 nitrogenous bases that bond with 3 complementary bases exposed (anticodon) on the tRNA opposite the attached amino acid
Wobble Aren’t 61 tRNAs, are 54tRNAs
Figure 17.4 The dictionary of the genetic code
Figure 17.3 The triplet code
tRNA complexes with its amino acid in the cytoplasm using ATP – activated tRNA
The activated tRNA-amino acid complex moves towards the ribosomal area and finds a triplet codon exposed that is complementary to the anticodon of the tRNA
The first activated tRNA-amino acid, after its anticodon is bound to the mRNA codon, associates with the large ribosomal subunit which now joins the smaller subunit and the mRNA and the tRNA (TAKE A BREATH!)
The first tRNA and its amino acid now occupy the P site of the large ribosomal subunit
Review – at this point the 2 part ribosome is assembled, the mRNA has started to be read, and one tRNA plus amino acid is occupying the P site
That means the adjacent A site is free to accept a second activated tRNA and its amino acid, but only if the anticodon of this tRNA matches the next three base pairs exposed (codon)
Protein Synthesis (continued)
At this point, there are 2 tRNA-amino acid complexes adjacent to each other – Elongation involved one amino acid being added in a three step process: Codon recognition – the mRNA codon in the A
site matches with the anticodon of the tRNA –amino acid complex
Peptide bond formation between the new amino acid in the A site and the amino acid (later peptide) in the P site
Translocation
Translocation – the ribosome moves the tRNA into the A site, and its attached peptide to the P site, as the previous tRNA from the P site moves to the E (Exit) site and leaves the ribosome
Review: once this process is under way, an activated tRNA with its amino acid finds an exposed codon in the A site, attaches via H-bonds, then forms a peptide bond with the polypeptide associated with the tRNA sitting in the adjacent P site. For a moment, the longer polypeptide chain is only attached to the tRNA in the A site. Now the entire ribosome shifts so that the………
Yet More Protein Synthesis The empty tRNA from the P site
moves in to the E site and leaves the ribosome
As the tRNA with the polypeptide chain moves from the A site to the now empty P site ….exposing a new codon. GUESS WHAT HAPPENS NEXT?!
A question? Every time a new codon is exposed in the
A site, a specific tRNA-AA complex moves into the site. What originally determined this mRNA Codon?
The Answer! The original DNA that was transcribed This elongation of 1 AA takes about 0.1 s Termination – the above continues
(dozens to hundreds or more AA added) until the STOP CODON is reached (codon at the end of the mRNA)
This codon does not have a matching tRNA anticodon so the tRNA-AA attaches in the A site and the tRNA moves to the E site and releases the polypeptide chain
FINALLY - SUMMARY The take home message:
At the ribosome, the genetic language of DNA is translated into a different language – Via RNA – into the functioning language of PROTEINS!!!!
Figure 17.17 The initiation of translation
Figure 17.18 The elongation cycle of translation
Figure 17.19 The termination of translation
Figure 17.20 Polyribosomes
Table 17.1 Types of RNA in a Eukaryotic Cell
Figure 17.23 The molecular basis of sickle-cell disease: a point mutation
Figure 17.24 Categories and consequences of point mutations: Base-pair insertion or deletion
Figure 17.24 Categories and consequences of point mutations: Base-pair substitution
Figure 17.25 A summary of transcription and translation in a eukaryotic cell
Figure 18.19 Regulation of a metabolic pathway
Control of Protein SynthesisRegulation of Gene Expression
Every cell has the same numbers and types of chromosomes
Development and normal gene function requires precise gene expression in an on and off manner
Operon – cluster of gene segments on DNA and its controlling segments Repressible Inducible
Regions of the Operon (DNA)Regions of the Operon (DNA)
Promoter region : promotes transcription by binding with RNA polymerase
Operator region : binds a regulatory protein or chemical Overlaps with the RNA polymerase
binding site Structural genes : code for a particular
peptide or several peptides Start or stop codes
Figure 18.20a The trp operon: REPRESSIBLE
Figure 18.21a The lac operon: INDUCIBLE
Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells