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From Flashcardlet > Genetics Test I > Flashcards

Genetics Test I Flashcards

1
Q

Semi conservative Replication

A

One strand of each new double helix is conserved from the parent molecule and the other is newly synthesized

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2
Q

Two stages for DNA replication

A

Initiation

  • Proteins open up the double helix.
  • Prepared DNA for base pairing

Elongation
-Proteins connect the correct sequence of nucleotides on newly formed DNA.

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3
Q

Why is DNA tightly regulated?

A
  • DNA replication is limited by DNA polymerase.
  • Can only add nucleotides in one direction, to the 3’ end
  • Leading strand grows continuously
  • Lagging strand grows only in a series of smaller Okazaki fragments
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4
Q

Initiation I

A
  • Initiation begins with double helix unwinding.

- Exposes the bases in the DNA strand.

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5
Q

Bidirectional Replication

A
  • When the DNA helix is opened up, goes in two directions
  • These sites are A-T rich. Have weaker interactions and so are easier to open up

DNA is bidirectional, this is what speeds it up.

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6
Q

Initiation II

A
  • Several proteins bind to the ori, forming a stable complex
  • Initiator protein binds to the origin of replication first.
  • DNA bound initiator attracts an enzyme called DNA helicase.
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7
Q

DNA helicase

A

Breaks apart the strands.

  • catalyses the localised unwinding of the double helix
  • creates two Y-shaped areas, one at either end of the unwound area (replication bubble)

DNA helicase can open up in both directions.

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8
Q

Replication Forks

A
  • Each Y is called a replication fork, consisting of two unwound DNA strands.
  • Single strand binding proteins stabilise the fork
  • Single strands serve as templates (molecular molds)
  • Formation of new DNA strand depends on the action of an enzyme DNA polymerase.
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9
Q

DNA polymerase III

A

Adds nucleotides. one after the other to the end of a growing DNA strand.

  • Only copies DNA that is unwound and maintained in the single stranded state
  • Adds nucleotides to the end of an existing chain
  • Functions in only one direction (5’ to 3’)
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10
Q

RNA Primer

A
  • Construction of a very short strand consisting of RNA nucleotides, provides link
  • Short stretch of RNA is called RNA primer.
  • Enzyme (primase) synthesizes RNA primer at the replication fork where base pairing takes place.
  • With double helix unwound and primer in place, DNA replication can take place in only one direction.

It is needed to start DNA replication

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11
Q

Role of RNA primers

A
  • Links together nucleotide subunits into a continuous strand of DNA
  • DNA polymerase catalyses the joining of nucleotides
  • Linkage of subunits through the formation of phophodiester bonds known as polymerisation.
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12
Q

Leading Strand

A
  • Helicase unwinds the double helix
  • DNA polymerase III moves in the same direction as the fork
  • Synthesis in 5’-3’ direction
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13
Q

Lagging strand

A
  • Strand is synthesised discontinually as small fragments (Okazaki fragments).
  • DNA polymerase III synthesises these small fragments in 5’-3’ direction
  • Each Okazaki fragment is initiated by a RNA primer
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14
Q

Okazaki fragment

A

-Polymerase adds nucleotides to the new primer crating an Okazaki fragment

DNA polymerase I- places RNA primers of the previously made Okazaki fragment with DNA

DNA ligase-covalently joins successive Okazaki fragments into a continuous strand of DNA.

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15
Q

Four general themes of Gene Expression

A
  1. Pairing of complementary bases
  2. Polarity (directionality) of DNA, RNA and proteins guide the mechanisms of gene expression.
  3. Requires input of energy and the participation of proteins and macromolecules
  4. Mutations change the information and therefore the phenotype.
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16
Q

Codon

A
  • Name given to each nucleotide triplet.

- All possible combinations of the four nucleotides in a codon, code for an amino acid

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17
Q

Gene transcription

A
  • Polymerisation of ribonucleotides guided by complementary base pairing, produces a RNA transcript of a gene.
  • Template is one strand of the DNA helix that composes the gene
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18
Q

Prokaryotes:

Initiation of transcription

A
  • Enzyme RNA polymerase catalyses transcription

- Promoter signals polymerase where to begin transcription

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19
Q

RNA polymerase

A
  • During initiation, the RNA polymerase consist of core enzyme, sigma subunit
  • Increase affinity of polymerase to the promoter, decreases affinity to DNA

RNA polymerase unwinds part of the DNA helix

  • aligns first two nucleotides of the new RNA at the 5’ end
  • Polymerase catalyses bond between them
  • RNA polymerase releases sigma unit
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20
Q

Prokaryotes:

Transcript elongation

A
  • sigma subunit loss, decreases affinity of polymerase to promoter, increases affinity for DNA.
  • Polymerase moves along chromosome, unwinding double helix (transcription bubble).
  • Extends RNA is the 5’-3’ direction moving along DNA in the 3’-5’ direction
  • Within the bubble, RNA hybridised to DNA
  • As DNA helix reforms, RNA strand displaced.
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21
Q

Prokaryotes:

Transcript Termination

A
  • Sequences in RNA signal the end of transcription (Termination)
  • Terminators often form hairpin loops
  • Polymerase and RNA strand released from DNA
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22
Q

Types of Terminators

A

Intrinsic- cause polymerase enzyme to terminate

Extrinsic- required other proteins to terminate

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23
Q

Prokaryotes:

Single stranded RNA produced

A
  • Known as ‘primary transcript’
  • Bases are complementary to the bases between the initiation/termination sites of gene DNA strand
  • RNA transcript carries bases for ‘Translation’ (Start/stop codon, codons specifying protein amino acids)
  • Transcript also called messenger RNA
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24
Q

Gene Expression

Prokaryotes vs. Eukaryotes

A

Prokaryotes
-Transcription of different genes may use different sigma factors

Eukaryotes

  • Promoters are usually more complicated
  • 3 kinds of RNA polymerase
  • Primary transcript is processed in nucleus, producing mRNA before protein synthesis.
25
Q

5’ end Methylated cap

A
  • 5’ end of eukaryotic mRNA is a guanine in reverse orientation
  • Backwards G not transcribe from DNA
  • Adding to transcript by a ‘capping enzyme’
  • Methyl group added to guanine by ‘methyl transferase’
  • Ensures mRNA’s stability for translation

Capping protects it from being broken down

26
Q

3’ end Poly-A tail

A

-3’ end of mRNA consists of 100-200 A’s (adenine)

Tail addition is a 3 step process

1) Ribonuclease cleaves primary transcript 3’ end
2) Enzyme ‘Poly-A polymerase’ adds A’s

27
Q

Splicing of Primary Transcript

A

DNA sequence of a gene is much longer than the mRNA.

Exons (Expressed regions)- sequences found in a genes DNA and mRNA

Introns (Intervening Regions)- sequences not found in a genes mRNA but DNA.

Prokaryotes, the gene has no interruptions

28
Q

Translation of mRNA

A
  • Takes place on ribosomes
  • Ribosomes coordinate the movement of tRNA, carry specific amino acids, one tRNA=one amino acid
  • mRNA provides the instructions for placing each tRNA.
29
Q

Transfer RNA

A

-Serves as an adaptor molecule
-Mediates transfer of information from RNA to protein.
Allows amino acids to join to the growing polypeptide.

-Short, single stranded RNA molecules

30
Q

Anticodon

A
  • Each tRNA carries an anticodon
  • Nucleotides are complementary to mRNA codon
  • Specifies the amino acid carried by the tRNA

tRNA anticodon 3’-5’
mRNA codon 5’-3’

31
Q

Aminoacyl tRNA synthetases

A
  • Enzyme helps amino acid attachment to tRNA
  • Enzyme is very specific
  • One enzyme for each amino acid
32
Q

Wobble

A
  • Some tRNAs can recognise more than one codon for amino acid they are charged with
  • Normal base pairing occurs at the first two positions
  • Wobble occurs at the third position.

Wasting resources if you make every single amino acid.

Inosine- modified adenine, can form base pairs with U and C

33
Q

Ribosomes in Polypeptide synthesis

A

Ribosomes facilitate synthesis by:

  • Recognise mRNA features that signal the start of synthesis
  • Supplies enzymatic activity that links amino acids to growing polypeptide.
  • Ensures linear addition of amino acids by moving 5’-3’ along mRNA sequences
  • Helps to end polypeptide synthesis by dissociating from mRNA and polypeptide
34
Q

Prokaryotic Mechanism of Translation

  1. Initiation
A

-Signal indicates where mRNA translation begins: Ribosome Binding Site

Site has two important elements

  • Shine Dalgarno box
  • 5’- AUG - 3’ triplet initiation codon.
35
Q

Initiation tRNA

A
  • Initiation tRNA with anticodon 3’-CAU-5’ binds to the initiation codon
  • It carries N-formylmethione (FMet)

16S rRNA binds to the Shine Dalgarno box

  • fMet tRNA binds to the initiation codon
  • fMet sits in P site of completed ribosome.

All prokaryotes begin with a modified fMet

36
Q

Eukaryotic differences Translation Initiation

A
  • Small ribosome binds to 5’ methylated cap
  • Migrates to first AUG
  • Initiator tRNA carries unmodified Met

We don’t need a modified methione of a shine delgarno box to initiate protein synthesis. Met sits in the P site.

37
Q

Elongation of Polypeptide

A
  • Elongation factor proteins direct the appropriate tRNA into the A site
  • Peptidyl transferase forms bonds between amino acids

Ribosome moves along to the next mRNA codon

  • Initiating tRNA transferred to the E site
  • Other tRNA carrying dipeptide to the A site
38
Q

Translation Termination

A
  • No tRNA for some codons (stop codons)-proteins recognise termination codon (Release factor)
  • tRNA releases completed polypeptide, tRNA and mRNA separate from ribosome and ribosome dissociates.
39
Q

Prokaryote Translation Summary

A
  1. Initiator tRNA carries Fmet.
  2. mRNAs have multiple binding sites and can thus direct the synthesis of several different polypeptides
  3. Small ribosomal subunits binds to the mRNAs ribosome binding site
40
Q

Eukaryote Translation Summary

A
  1. Initiator tRNA carries methione
  2. mRNAs have only one start site, direct the synthesis of only one kind of polypeptide
  3. Small ribosomal subunits bind first to the methylated cap at the 5’ end.
41
Q

Regulation of Gene Expression

A

How much protein made at any time is under different controls

Controls affecting transcription

  • binding of the RNA polymerase to promotor
  • shift from transcription initiation to elongation
  • release of mRNA at transcription termination

Controls post transcriptional

  • determine mRNA stability after synthesis
  • stability of the polypeptide product
42
Q

Operon Theory

A
  • A single signal can regulate the expression of several genes
  • Genes clustered together on chromosome and involved in the same response. Under the control of a single promoter
  • Genes can be transcribed together into a single mRNA
  • Cluster of genes regulated in this way are called ‘Operons’
43
Q

Lactose (lac) operon

A

Single DNA unit enabling simultaneous regulation of 3 structural genes.

lac Z, Y, A.

44
Q

Induction/Repression of Lac Operon

A

Repression

  • No lactose, repressor binds to the operon
  • negative regulatory element

Induction

  • Lactose present, allolactose binds the receptor
  • Repressor protein changes conformation, unable to bind to operator
  • RNA polymerase can gain access to the lac operons
45
Q

cAMP Receptor Proteins (CPR)

A
  • A small nucleotide cAMP binds to CRP
  • Enables CRP to bind to regulatory region of the lac operon
  • Increases RNA polymerase ability to transcribe

Glucose indirectly control amount of cAMP

  • Glucose absent, cAMP is high
  • Glucose present, cAMP is low.
46
Q

araC as a Positive Regulator

A
  • araC is a positive regulatory protein specific for all the arabinose genes (responsible for the breakdown of sugar arabinose)
  • Arabinose genes (araB, A, D) arranged in an operon, induced when arabinose is present
  • Arabinose binds to araC, conformational change of protein, araC binds to next promotor, allowing transcription
47
Q

How Attenuation works

A

When tryptophan present:

  • Ribosome moves quickly past trp codon.
  • Prevents formation of loop between 2-3

Absent:

  • Ribosome stalls at the trp codon
  • Allows formation of loop between 2-3
48
Q

Basal factors

A

Assist the binding of RNA polymerase II to the promoter. Initiates low level transcription called basal transcription.

-Key component: TATA box binding protein.

49
Q

Basal Transcription

A
  • Associates with several other basal factors called TBP associated factors (TAF)
  • TBP binds to the TATA in the promoter DNA
  • 3 TAFs bind to TBP
  • RNA pol II binds to these basal factors
50
Q

Enhancer elements

A
  • When bound, transcription factors can interact directly or indirectly with the promotor basal factors.
  • Causes an increase in transcriptional activity
51
Q

Activator Protein

A

Binds to enhancer, increasing transcriptional activity.

Activator proteins bound to DNA, increase RNA synthesis via 3 ways:

1) Stimulate ‘recruitment’ of the basal transcription machinery (RNA pol II, TBP, TAF)
2) Stimulate activity of the basal factors already bound to the promotor
3) Facilitate the changes in chromatin structure.

52
Q

Activator Domains

A

To function, activator proteins must have two structural domains.

  • Bind to enhancer DNA in a sequence specific way= DNA binding domain
  • Be able to interact with other proteins= Transcription Activator Domains.

A small number of protein motifs appear in DNA binding domains of different activator proteins.

Best characterised are the:

  • Helix loop helix and the helix turn helix motif
  • Zinc finger motif
53
Q

DNA binding Domain Motifs

A
  • They form grooves which allows DNA to interact with them
  • The function of each motif is to promote binding to the DNA double helix
  • The protein fits within or interacts within the major groove of DNA

e.g. Steriod hormone receptors

54
Q

Coactivators

A

Proteins and other molecules that play a role in transcriptional activation without binding directly to DNA.

55
Q

Formation of Dimers

A

Many transcription regulators are multimeric proteins.

Homomers- identical protein subunits, Jun-Jun
Heteromers- non identical protein subunits, Jun-Fos

Each of these dimers recognise different enhancer sequences

56
Q

Dimerisation Domain

A

Dimerisation occurs through a transcription factor domain

  • Dimerisation domain
  • Allow specific protein to protein interaction
  • Certain motifs recur in dimerisation domains

Most common in the leucine zipper motifs

  • Helix with leucine residues at regular intervals
  • Leucine zipper motif interlocks like a zipper with a leucine zipper motif on another polypeptide

Once a dimer is form, it can interact with DNA.

57
Q

Repressors

A

Transcription factors that suppress transcription caused by activator proteins.

Different receptors can act in different ways:

  • compete with activator proteins for binding to the same enhancer (competition)
  • Binds directly to a specific activator (Quenching)
  • Acts directly o the promotor to eliminate almost all transcriptional activity.

Type I- repressor binds to and blocks the DNA binding region of an activator
Type II- Repressor binds to and blocks the activation domain of an activator.

58
Q

Myc-Max control of cell proliferation

A
  • Myc polypeptide contains an activation domain, alone it cannot bind DNA and act are an activator
  • Max polypeptide binds to DNA, alone has no activation domain so cannot act as a activator when bound to DNA

Only when Myc-Max come together, it will carry out the activity.

59
Q

What are the main cis and trans regulatory elements found in the E. coli lac operon and describe how they regulate the expression of genes important for lactose utilization.

A

Cis
-Promoter site: RNA polymerase binds and initiates transcription
-Operator: site near the promoter on the same DNA molecule
-Both act cis
Affect the expression of downstream structural lac genes on the same DNA molecule

Trans
Repressor proteins
Binds to the operator
Encoded by the lacI gene which is separate from the operon and unregulated. Produced all of the time
-After synthesis, the repressor diffuses through the cytoplasm and binds with its target.
An inducer prevents binding of the repressor to the operator

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