1
Q
Gene
A
- unit of heredity
- not a base pair, not a defined length
- genes contain the information necessary to make an organism and allow it to interact with its environment
- segments of DNA that code for something
- most genes code for polypeptides; a lot of “junk” DNA though: not genes
- polypeptides become a unit of function or protein
- activities of proteins determine structure and function of cell
- traits of characteristics of an organism- based on cellular activities
2
Q
human genome
A
- collection of all genes needed for a human
- just expressed differently
3
Q
gene expression
A
- gene function at the molecular level
- gene function at the level of traits
- the two levels are tightly woven together
- gene expression affects structure and function of cell-> determines traits of an organism
4
Q
fly experiment
A
??
5
Q
how does the cell read the information contained in the genomic DNA?
A
- DNA=linear polymer of nucleotides
- the order of the nucleotides carries the information needed to make gene products
- two step process 1. transcription and 2. Translation
6
Q
What makes up a gene?
A
- region of DNA, 100’s to 1,000’s bp long
- promoter
- Transcribed region aka “coding region”
- Terminator
7
Q
beginning with the simplest level of structure, which order of organization of genetic material is correct?
A
nucleotide, DNA, gene, chromosome and genome
8
Q
Mendel
A
-had the idea that genetic information is transmitted by a particle carrying genetic information
9
Q
wild type
A
- has nothing done to it
10
Q
Archibold Garrod
A
- postulated that genes coded for enzymes
- inherited disease might be due to missing gene/ enzyme
11
Q
Garrod studied Alkaptonuria
A
- Phenylalanine= essential amino acid
- Cannot be made, must be consumed
- in excess, it is converted to tyrosine and eliminated
- conversion to tyrosine involves multiple pathways
12
Q
Beadle and Tatum studied Neurospora crassa
A
- common bread mold
- minimal requirements for growth are carbon source (sugar), inorganic salts and the vitamin biotin
- N. crassa has all the enzymes necessary to make small molecules (amino acids, vitamins) for survival and growth
- studied mutant strains of mold
- strains defective in amino acid synthesis
- Arginine auxotrophs- can’t make arginine
- unable to grow unless food is supplemented with arginine
- created mutants by exposing mold strains to X-rays
- concluded that a single gene controls the synthesis of a single enzyme
- one gene- one enzyme hypothesis
- close, not right
13
Q
One Gene One enzyme?
A
- one gene- one enzyme hypothesis has been modified
- all genes encode proteins, not all proteins are enzymes
- more accurate: one gene encodes a polypeptide
- ex: hemoglobin composed of 4 polypeptides required for function
- 2 a polypeptides from one gene
- 2 B polypeptides from another gene
- one gene- one polypeptide theory
14
Q
Central dogma
A
-DNA to mRNA to protein?
15
Q
Transcription
A
- copying DNA into an RNA molecule or transcript
- DNA stays in nucleus
- making photocopies of book pages in the library
- the copies CAN leave the library
- RNA polymerase= the copy machine
- over 90% of all genes are structural
- directions for making a polypeptide
- produce a copy known as mRNA
- mRNAs carry the genetic information to the ribosomes
- where are ribosomes located?
16
Q
3 stages of Transcription
A
- initiation
- elongation
- termination
17
Q
Promoter; transcription
A
needed to initiate transcription
18
Q
Terminator; transcription
A
needed to end transcription
19
Q
Direction of transcription
A
- direction of transcription and DNA strand used varies among genes on a given chromosome
- in all cases, synthesis of RNA transcript is 5’ to 3’
- DNA template strand is always read 3’ to 5’
20
Q
eukaryotic transcription
A
- basic features identical to prokaryotes
- however, each step has more proteins
- greater complexity and regulation
- 3 forms of RNA polymerase in eukaryotes
- RNA polymerase II- transcribes mRNA
- RNA polymerase I and III- transcribes nonstructural genes for rRNA and tRNA
21
Q
RNA processing
A
- bacterial mRNAs- translated into polypeptides immediately
- Eukaryotic mRNAs
- initial long pre-mRNAs processed -> mature mRNA
- Introns- transcribed but not translated -> removed before export to cytosol
- Process:
- 7 methyl G cap addition to 5’ end
- splicing- removal of introns and connection of exons
- PolyA tail added to 3’ end
22
Q
Capping (RNA processing)
A
- 7 methyl G cap addition to 5’ end
- needed for proper exit of mRNA from nucleus and binding to ribosome
23
Q
tailing (RNA processing)
A
- adding a poly A (adenine) tail
- increases stability and lifespan in cytosol
24
Q
introns vs. exons
A
- exons- parts of DNA that are translated
- introns- parts of DNA that are not translated
25
Splicing- Removal of introns
- introns found in many eukaryotic genes
- widespread in complex eukaryotes
- human dystriphin has 79 exons and 78 introns
- introns are rare in all prokaryotes
- introns removed from eukaryotic pre-mRNA by a large complex called spliceosome
- composed of subunits called snRNPs (snurps)
- Small Nuclear RNA and a set of Proteins
26
Steps of Splicing- Removal of Introns
- intron RNA defined by particular sequences
- e.g 5' splice site, branch site, 3' splice site
1. Spliceosome subunits recognize intron sequences
2. binding causes intron to loop outward
3. two exons brought closer together
4. 5' splice site cut first -> 5' end of intron binds to branch site -> 3' splice site cut
5. 5' and 3' ends are covalently bonded
27
Alternative splicing
- function of spliceosome regulated so that single gene can encode 2 or more polypeptides
- no longer "one gene- one polypeptide"
- only higher organisms demonstrate alternative splicing
- contributes to greater complexity of life
- this is how a human can have 25,000 genes BUT make 100,000+ proteins
28
what involves a promoter region?
initiation of transcription
29
translation
- interpretation
- of mRNA nucleotide language into the amino acid language of proteins
- tRNA= translator, Ribosome's= mediator
- mRNA read in codons
- 64 codons (4 bases in groups of 3)
- 20 amino acids -> some amino acids encoded by >1 codon
- 1 start, 3 stop codons
- degenerate- more than one codon for same amino acid
30
bacterial mRNA
- 5' ribosomal-binding site (Shine-Delgarno)
- start codon usually AUG
- 1 of 3 stop codons
- UAA, UAG or UGA
31
anticodon
allows binding of tRNA to mRNA codon
32
reading frame
- start codon defines reading frame
- small changes in the DNA sequence can have major effects
- addition of a U shifts the reading frame
- can cause the production of a completely different protein
- look at chart
33
Requirements of Translation
-mRNA, tRNA, ribosome's, translation factors and requires a lot of energy
34
tRNA
- amino acid attachment site is at the 3' single-stranded region
- different tRNA molecules encoded by different genes
- E.g., tRNAser carries serine
- common features
- cloverleaf
- anticodon
- acceptor stem for amino acid binding
35
aminoacyl-tRNA synthetase
- catalyzes the attachment of amino acids to tRNA
- one for each of the 20 different amino acids
- considered the second genetic code
36
Ribosomes
- prokaryotes have one kind
- Eukaryotes have distinct ribosomes in different cellular compartments
- Mitochondria, chloroplast and cytosol
- think back to symbiosis theory
- focus on cystolic ribosomes
- large and small subunits
- structural differences exploited by antibiotics
- inhibit bacterial ribosomes only
37
S terminology
the Svedberg unit measures particle size based on its rate of travel in a tube subjected to high g-force
38
Ribosome shape
- determined by rRNA
- discrete sites for tRNA binding and polypeptide synthesis
- P site- peptidyl site
- A site- aminacyl site
- E site- exit site
39
Stages of Translation
1. initiation- mRNA, first tRNA an ribosomal subunits assemble
2. Elongation- synthesis from start codon to stop codon
3. termination- complex disassembles at stop codon releasing completed polypeptide
40
Translation: Stage 1: Initiation (bacteria)
- mRNA binds to small ribosomal subunits facilitated by ribosomal-binding sequence
- start codon a few nucleotides downstream
- initiator tRNA recognizes start codon in mRNA
- large ribosomal subunit associates
- at the end, the initiator tRNA is in the P site
41
Translation: Stage 1: Initiation (eukaryotic)
- instead of a ribosomal-binding sequence, mRNAs have a 7-methylguanosine cap at 5' end
- recognized by cap-binding proteins
- position of start codon more variable
42
Translation: Stage2: Elongation
1. aminoacyl tRNA brings a new amino acid to the A site
- binding occurs due to codon/anticodon recognition
- two types of tRNA:
1. Peptidyl tRNA: tRNA with a peptide chain
- occurs in the P site of ribosomes
2. Aminoacyl tRNA: tRNA with an amino acid
- occurs in the A site of ribosomes
43
Translation: Stage 3: Termination
- when a stop codon is found in the A site, translation ends
- 3 stop codons- UAA,UAG,UGA
- recognized by release factors
- completed polypeptide attached to a tRNA in the P site and stop codon in the A site
- release factor binds to stop codon at the A site
BIO 111
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