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Diana's Flashcards > genetics exam 3 > Flashcards

genetics exam 3 Flashcards

1
Q

what is the conservative model?

A

both parental strands stay together after DNA replication

2nd generation contains separate 15N and 14N

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2
Q

what is the semiconservative model?

A

the dsDNA contains one parental and one daughter strand after replication

2nd generation contains hybrid of 15N /14N and a separate 14N

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3
Q

what is the dispersive model?

A

parental and daughter DNA segments are interspersed in BOTH strands after replication

2nd generation only contains hybrid of 15N/14N

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4
Q

which DNA label is light and heavy?

A

15N = heavy media
14N = light media

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5
Q

how are nucleotides joined?

A

via phosphodiester linkages

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6
Q

which end has the hydroxyl and the phosphate?

A

5’ P
3’ OH

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7
Q

what direction does DNA polymerase catalyze polymerization?

A

5’-3’ (DNA template is 3’-5’)

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8
Q

what does DNA polymerase require to polymerize?

A

it cannot initiate DNA synthesis by linking two individual nucleotides together and can ONLY add onto PRE-EXISTING 3’ OH end of DNA or RNA

it requires a starting piece of RNA primer at the 3’ OH end (added by RNA polymerase)

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9
Q

what directions do the leading and lagging strands synthesize DNA in?

A

leading strand = DNA pol III attaches to the nucleotide in 5’-3’ and slides toward the OPENING of the replication fork

lagging strand moves AWAY from the replication, DNA pol III also attaches in 5’-3’ but contain okazaki fragments which will be sealed by DNA ligase

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10
Q

how many primers are needed in the leading and lagging strands?

A

leading = one primer
lagging = multiple primers

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11
Q

where does DNA synthesis begin in bacteria?

A

at site called oriC

each bacterial chromosome only has ONE origin of replication and proceeds BIDIRECTIONALLY

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12
Q

when does bacterial DNA synthesis end?

A

two replication forks will eventually meet at the opposite side of the chromosome, which ends the replication

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13
Q

what are the 3 types of DNA sequences in oriC that are functionally significant?

A

AT-rich region - where the two strands separate when the DnaA proteins bind to the DnaA boxes

DnaA boxes - DnaA proteins (help with bending of the chromosome) bind to DnaA boxes

GATC methylation sites - regulates replication , DNA adenine methyltransferase methylates A on both strands (immediately after replication, daughter strands are unmethylated, both the dsDNA is hemimethylated due to the parental strand)

INITIATION OF REPLICATION ONLY OCCURS EFFICIENTLY ON FULLY METHYLATED DNA

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14
Q

what is DnaB?

A

it is the bacterial chromosomal helicase that unwinds the dsDNA and replicates in 5’-3’ CW and CCW (bidirectional), using energy

bacterial replication contains TWO REPLICATION FORKS

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15
Q

what is the function of topoisomerase II?

A

aka DNA gyrase which travels ahead of helicase and alleviates the supercoils

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16
Q

what is the function of SS binding proteins?

A

bind to the separated DNA to keep them separated

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17
Q

what are the functions of DNA pol I and III?

A

involved in normal DNA replication

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18
Q

what is the function of DNA pol II, IV, and V?

A

needed for DNA repair and replication of damaged DNA

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19
Q

how does DNA pol III contribute to bacterial DNA replication?

A

it synthesizes the daughter strand

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20
Q

how does DNA pol I contribute to bacterial DNA replication?

A

removes RNA primers and replaces the RNA with DNA using 5’-3’ exonuclease activity to digest the RNA and a 5’-3’ polymerase to replace it with DNA

also has 3’-5’ exonuclease activity so that it can go backwards to correct its mistakes

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21
Q

how does DNA ligase contribute to bacterial DNA replication?

A

it seals the gaps in the sugar-phosphate backbone

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22
Q

what are the multiple proteins found at the replication fork called?

A

replisome complex

the 2 DNA pol II (on the leading and lagging strand) move as a unit during replication –> allows the coordination of leading and lagging strand synthesis

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23
Q

what is significant about lagging strand DNA synthesis?

A

the lagging strand is LOOPED –> allows DNA pol II to synthesize the okazaki fragments in 5’-3’ and move as a unit with the leading strand DNA pol III

completion of okazaki fragment –> enzyme releases lagging template strand, clamp loader complex reloads pol at next RNA primer and another loop is formed –> process repeats

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24
Q

what are the subunits of DNA pol III called?

A

DNA pol III holoenzyme

synthesizes DNA 5’-3’, 3’-5’ proofreading, clamp protein (allows DNA pol to slide along DNA without falling off - maintain association of DNA with pol II), clamp loader complex (helps clamp protein bind to DNA, uses ATP hydrolysis to open beta clamp and close it around template DNA)

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25
Q

what is on the opposite of oriC?

A

a pair of termination sequences aka TER SEQUENCES (T1 stops CCW and T2 stops CW)

Tus binds to ter sequences and stops the movement of the replication forks

only ONE ter sequence is required to stop one fork and the other fork ends its DNA synthesis when it reaches the stopped fork

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26
Q

what are the intertwined circular molecules at the end of bacterial DNA replication called?

A

catenanes which will be separated by topoisomerase II (used to prevent supercoils in dsDNA)

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27
Q

what type of chromosomes do eukaryotes have?

A

long, linear chromosomes
require multiple origins of replication

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28
Q

what is unique in the replication of eukaryotic chromosomes?

A

they form replication bubbles from multiple origins that merge into replicated chromosomes (begins and ends in S phase)

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29
Q

what is the origin of replication in S. cerevisiae called?

A

ARS elements
have high percentage of A and T
ARS consensus sequences (ATTTAT - A or G - TTA)

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30
Q

explain eukaryotic replication

A

begins with assembly of prereplication complex (preRC) which includes the origin recognition complex (ORC) which acts as the FIRST INITIATOR of eukaryotic DNA replication

other preRC proteins = MCM helicase, binding of MCM completes DNA replication licensing

activates in S PHASE, origins are ABLE TO BEGIN DNA SYNTHESIS

G1 phase = ORC binds to origin, other preRC assemble on origin, MCM binds to leading strand

S phase = preRC converted to active replication site by phosphorylation, MCM moves in 3’-5’ and DNA replication occurs bidirectionally

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31
Q

what are the function of the different eukaryotic DNA polymerases?

A

alpha, delta, and epsilon = nuclear DNA
gamma = mitochondrial DNA

alpha = polymerizing
beta = repair

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32
Q

what are the functions of delta and epsilon DNA pol?

A

delta = replication of LAGGING strand
epsilon = replication of LEADING strand

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33
Q

what are some differences between DNA replication in bacteria vs. eukaryotes?

A

bacteria = polymerases are also EXONUCLEASES, one origin of replication, Okazaki fragments 1000-2000

eukaryotes = not all polymerases are exonucleases, many origins of replication, Okazaki fragments 150-200, histones complexed to DNA

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34
Q

what is the function of DNA pol alpha?

A

associated with PRIMASE (10 RNA nucleotides followed by 20-30 DNA nucleotides)

exchange of DNA pol alpha for epsilon or delta is required for the elongation of leading and lagging strands = polymerase switch

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35
Q

what is the function of DNA pol in DNA repair?

A

DNA pol beta is NOT involved in DNA replication
plays role in removal of incorrect bases from damaged DNA

many are TRANSLESION-REPLICATING POLYMERASES (replication of damaged DNA, can synthesize a complementary strand over abnormal region)

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36
Q

what is the function of flap endonuclease?

A

removes RNA primers

polymerase delta runs into primer of adjacent okazaki fragment –> pushes portion of RNA primer into short flap and FLAP ENDONUCLEASE removes it

if flap is too long, it is cleaved by Dna2 nuclease/helicase (cuts long flap into short flap)

process continues until the entire RNA primer is removed and DNA ligase seals the two fragments together

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37
Q

what problem arises at eukaryotic chromosomal ends?

A

lagging strands CANNOT be replicated at 3’ end which can lead to chromosome shortening because unreplicated sequences at ends get shorter and shorter

also loss of important DNA sequences from ends of chromosomes –> problem resolved by adding telemores

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38
Q

what is the function of telomerase?

A

extends the ss PARENTAL template strand, lagging strand synthesis makes the complementary copy

contains protein and RNA

RNA is complementary to DNA sequence in telomeric repeat which allows telomerase to bind to 3’ overhang via RNA-DNA base pairing, rest of RNA acts as a template for addition of DNA to chromosome end

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39
Q

why are telomeres important?

A

linear eukaryotic chromosomes have telomeres at both ends

they are complex of telomeric DNA sequences and bound proteins which consist of moderately repetitive tandem arrays, ending in 3’ overhang (12-16 nt long)

telomeric sequences consist of several GUANINE nt and many THYMINE nt

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40
Q

what is the function of reverse transcriptase activity?

A

can copy RNA into DNA (normally you copy DNA into RNA to make mRNA)

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41
Q

describe the enzymatic steps of telomerase

A
  1. telomerase binds to the 3’ overhand
  2. telomerase synthesizes 6-nt repeat
  3. telomerase moves 6 nt to the right and begins making another repeat

steps are repeated many times to lengthen one strand of chromosome and other strand is extended by lagging strand synthesis using primer and DNA pol and ligase

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42
Q

what is the telomeric nt sequence in eukaryotes?

A

TTAGGG

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43
Q

what is TERT?

A

telomere reverse transcriptase
protein subunit of telomerase that is an RNA-dependent DNA polymerase

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44
Q

what cells express telomerase?

A

expressed only in embryo
tend to shorten in actively dividing cells
can shorten to 1,500 in elderly person

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45
Q

what is senescent?

A

cells become senescent when telomeres are short (stop dividing/growing)

insertion of highly active telomerase can block senescence

cancer cells carry mutations that increase activity of telomerase which prevents the telomere from shortening and senescence

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46
Q

what can mutations in genes involved in telomere function lead to?

A

genetic disorders like Werner syndrome or dyskeratosis congenita

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47
Q

what can the rate or telomere shortening tell you?

A

it is a good predictor of life span for different species (faster rate = shorter lifespan)

mean telomere length at birth is NOT a good indicator of lifespan

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48
Q

DNA base sequences define…

A

the beginning and end of a gene and regulate the level of RNA synthesis

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49
Q

control of gene expression occurs at?

A

initiation of transcription

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50
Q

what determines whether a gene will be transcribed?

A

regulatory proteins which are usually right next to the promotor (where RNA pol binds)

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51
Q

what is the function of regulator sequences?

A

DNA sites for the binding of regulatory proteins

role of regulatory proteins is to INFLUENCE RATE OF TRANSCRIPTION

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52
Q

what is the function of transcription factors?

A

proteins that recognize the promoter and regulatory sequences to control transcription

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53
Q

what does polycistronic mean?

A

bacterial mRNA may be polycistronic which means it encodes two or more polypeptides - a single mRNA may encode multiple proteins

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54
Q

the RNA transcript is complementary to…

A

the template strand

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55
Q

what are the stages of transcription in bacteria?

A
  • initiation: promoter functions as a recognition site for RNA pol (RNAP), after binding –> DNA is denatured into a bubble known as open complex
  • elongation/synthesis of the RNA transcript: RNA pol slides along the DNA in an open complex to synthesize RNA
  • termination = terminator is reach that causes RNA pol and RNA transcript to dissociate from the DNA
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56
Q

what are examples of functional RNAs?

A

tRNA, rRNA
RNA components of:
- spliceosomes
- signal recognition particles
- telomerase (composed of RNA and proteins)
small regulatory RNAs

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57
Q

where are promoters located?

A

upstream of site where transcription of a gene begins

bases in a promoter sequence are numbered in relation to the transcription start site (+1)

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58
Q

what are consensus promoter sequences?

A

sequences in the -35 and -10 sequences in the promoters of many different genes that will be conserved if they are important

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59
Q

what does the RNA pol holoenzyme consist of?

A

core enzyme (makes the RNA, binds weakly to DNA and transcribes non-specifically) and a sigma factor (recognizes the -10 and -35 sequences)

altogether, holoenzyme can recognize promoters with specificity and transcribe DNA

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60
Q

how is transcription initiated in bacteria?

A

binding of RNA polymerase at promoter forms CLOSED COMPLEX (dsDNA)

OPEN COMPLEX is formed when TATAAT box in -10 region is unwound (ssDNA)

short RNA strand is made in the open complex, sigma factor is released at this point which marks the end of initiation

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61
Q

what happens to the DNA behind the open complex?

A

DNA rewinds back into a double helix

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62
Q

what is the rate of RNA synthesis?

A

43 nt per second

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63
Q

explain RNA synthesis

A

RNA pol slides along DNA, creating an open complex –> template DNA is used to make a complementary RNA-DNA hybrid –> RNA pol moves along template in 3’-5’ and RNA is synthesized in 5’-3’ (does NOT need primers)

TEMPLATE DNA STRAND AND mRNA ARE ANTIPARALLEL (mRNA is the same as the parental strand)

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64
Q

which ends are the promoters and terminators located?

A

promoters are always at the 3’ end
terminators are always at the 5’ end

65
Q

what are the two different mechanisms that E. coli use for termination of transcription?

A

rho-dependent termination (REQUIRES protein known as r - rho) –> termination factor Rho catches up with RNAP at secondary structure and unwinds RNA from the hybrid in the transcription bubble, releasing RNA

rho-independent termination (DOES NOT require r) –> Rho-independent termination/intrinsic termination is facilitated by two sequences in the RNA
- URACIL RICH SEQUENCE located at 3’ end
- G-C RICH STEM-LOOP upstream of uracil rich sequence

66
Q

how does eukaryotic RNA pol differ from bacterial RNA pol?

A

eukaryotes have 3 RNA pol while bacteria only have ONE

RNA pol I = transcribes all rRNA genes (except for 5S rRNA) –> these are transcribed as a single big RNA and then cut into pieces

RNA pol II = transcribes all PROTEIN-ENCODING gene (all mRNAs) and some snRNA genes needed for SPLICING

RNA pol III = transcribes all tRNA genes, 5S rRNA gene, microRNA genes

67
Q

what are the eukaryotic promoter sequences?

A

they have a core promotor (important in determining precise start point for transcription) which consists of:
- TATA box (located in -25 sequence)
- transcriptional start site or initiator

and regulatory elements

CORE PROMOTER PRODUCES BASAL LEVEL OF TRANSCRIPTION

68
Q

what are some factors that can affect eukaryotic transcription?

A

promoter-proximal elements
enhancers (stimulate transcription)
silencers (inhibit transcription)

both enhancers and silencers are often found in the -50 to -100 region

69
Q

how does eukaryotic transcription differ from bacterial?

A

RNA pol II transcribes all pre-mRNAs
contains general transcription factors (GTFs) and a mediator (mediates interactions between RNA pol II and regulatory transcription that bind enhancers or silencers)

RNA pol (I, II,III) CANNOT bind directly to a promoter, there is NO sigma subunit –> instead GTFs first bind to the promoter and recruit RNA pol

(LECTURE 25, SLIDE 6)

70
Q

explain the process of transcription in eukaryotes

A

TFIID binds to the TATA box (TFIID includes TATA-binding protein - TBP and what TBP associated factors - TAFs) –> TFIIB binds to TFIID, promoting the binding of RNA pol II to core promoter, TFIIF is bound to RNA pol II –> TFIIE and TFIIH bind to RNA pol II and form a preinitiation/closed complex –> TFIIH acts as a helicase to form an open complex and it also phosphorylates C-terminal domain of RNA pol II which helps release all the GTFs

in order for RNA pol II to leave, the c-terminal domain has to be PHOSPHORYLATED

71
Q

how does transcriptional termination in eukaryotes differ from bacteria?

A

mRNAs in eukaryotes have POLY A TAIL added at 3’ end (RNA pol II transcribes the polyA signal into AAUAAA in RNA)

pre-mRNAs are then cleaved downstream of this signal, transcription terminates about 500-2000 nt downstream from polyA signal

71
Q

what is colinearity?

A

sequence of DNA in non-template strand corresponds to sequence of nt in mRNA

sequence of codons in mRNA provides instructions for sequence of AA in polypep

72
Q

do eukaryotic structural genes display colineraity?

A

not always
DNA sequences were found to be much larger than corresponding mRNAs because of R looping –> mRNA is interrupted by DNA loops which contain sequences only present in DNA and not mRNA

73
Q

what does the pre-mRNA sequence consist of?

A

both introns and exons
introns will be later be removed via splicing and the exons will connect together to make mature mRNA

74
Q

explain what is splicing in eukaryotic genes

A

initial transcript known as mRNA contains introns and exons which spliceosomes will splice before it can leave the nucleus

spliceosome is composed of snRNPS - each contains small nuclear RNA and a set of proteins
each subunit carries out different functions
- bind to intron seuqnece and precisely recognize the intron-exon boundaries
- hold the pre-mRNA in the correct configuration
- catalyze the chemical reactions that remove introns and covalently link exons

75
Q

how does splicing occur?

A

intron contains seuqneces for splicing
- GU at 5’ end and AG at 3’ end
- internal BRANCHPOINT which contains A –> essential

U1 binds to the 5’ splice site (GU) and U2 binds to branch site (branchpoint A) –> U4/U6 and U5 trimer binds, looping intro out and exons are brought closer together –> 5’ splice site is cute, 5’ end of intron is connected to A, (FORMS A LARIAT or lasso) U1 and U4 are released –> 3’ splice site is cut, exon 1 is connected to exon 2, intron is released with U2, U5, and U6

INTRON IS DEGRADED

76
Q

where does splicing occur?

A

in the NUCLEUS as RNA is being transcribed

77
Q

what can some introns do?

A

they have the ability to self-splice
meaning intro removal in absence of snRNPs/factors, catalytic power is provided by RNA sequences in the intron

Group I and II introns
I = initiated by GTP
II = same steps as pre-mRNA with lariat intermediate (lasso)

78
Q

what is alternative splicing?

A

pre-mRNAs may be spliced in many ways, creating different combos of exons that can be joined together to produce different mRNAs (can have exon skipping)

allows DIFFERENT polypeptides to be made from the SAME GENE (# of proteins produced in a cell may far exceed number of protein-coding genes in that cell)

79
Q

how can RNA transcripts be modified?

A

final RNA differs from the initial transcript

mature mRNAs have 7-methyl-guanosine at their 5’ end aka CAPPING –> occurs as the pre-mRNA is being synthesized by RNA pol II aka co-transcriptional (cap-binding proteins recognize CAP structure on RNA, CAP is important for movement of some RNAs into the cytoplasm and INITIATION OF TRANSLATION OF mRNA)

mature mRNAs also have polyA tail which is NOT ENCODED in gene sequence, added after gene is completely transcribed, polyA tail is important for translation, export, and mRNA stability

80
Q

what is the order of co-transcription of pre-mRNA?

A

initiation of transcription
addition of 5’ cap
splicing
addition of polyA tail
transport out of nucleus into cytoplasm

81
Q

what is RNA processing?

A

cleavage of large RNA transcript into smaller pieces, one or more of the smaller pieces becomes a functional RNA molecule

  • many nonstructural genes are initially transcribed as large RNA –> large RNA transcript is cleaved into smaller pieces
  • transfer RNAs are also made as large precursors that will be cleaved at 5’ and 3’ ends to produce mature, functional tRNAs
82
Q

what is the large rRNA transcript made by?

A

made by RNA pol I

large ribosomal subunit contains 5S, 5.8S and 28S
small ribosomal subunit contains 18S

83
Q

explain processing of a precursor tRNA molecule

A

RNaseP (endonuclease) makes cuts in the tRNA sequence to remove unnecessary sequences and generate a mature molecule

RNaseD (exonuclease) removes nucleotides from the ends of the molecule

84
Q

what is RNA editing?

A

change in the nt sequences of an RNA
can involve addition or deletion of bases
only a FEW SPECIFIC based in a FEW mRNAs are targeted for editing

ex: deamination converts RNA nt to new forms (cytosine to uracil, adenine to hypoxanthine)

another kind of editing uses small guide RNAs to add/remove bases

85
Q

what are some examples of RNA editing?

A

trypanosome - primarily addition but sometimes deletion of one or more URACILS

slime mold - CYTOSINE additions

mainly cytosine to uracil and adenine to hypoxanthine

86
Q

what are the arginine analogs?

A

ornithine and citrulline

87
Q

explain genetic analysis in relation to arginine

A

if an arg- mutant grows on an intermediate, the block in pathway affected by the mutation is before the intermediate is made

ex: arg-2 can grow in citrulline so if you supply it with citrulline it can convert it to arginine but it CANNOT convert ornithine to citrulline

if a mutant grows on a particular compound but not on MM, the block is BEFORE synthesis of that compound –> the more compounds a mutant grows on, the earlier the pathway block is

88
Q

the reason why Beadle and Tatum observed four different categories of mutants that could not grow on media without methionine is because

A

four different enzymes are involved in a pathway for methionine biosynthesis

89
Q

what is the one gene-one enzyme hypothesis?

A

a single gene controlled the synthesis of a single enzyme

ex: normal strains = met was synthesized by cell enzymes but in mutant strains = genetic defect in one gene prevented the synthesis of one protein required in one step of the pathway to produce that AA

90
Q

how can you determine the order in which compounds are produced?

A

earlier intermediates in the pathway = fewer number of mutants that will grow on it

later intermediates in the pathway = more mutants will grow on it

order of genes are decreasing in number

91
Q

what types of frameshift will result in mutant vs. WT?

A

mutant = 1,2,4,5 deletions or additions

WT = 1 deletion and 1 addition (cancel each other out), 3 or 6 deletions or additions

92
Q

how was the triplet code determined?

A

used phage T4 rLLB gene as an experiment
lots of +1 or -1 mutations available
these are readily combined by recombination

insertions or deletions of groups of 3 nt DON’T shift the reading frame

93
Q

what are other characteristics of the genetic code?

A

it is NON-OVERLAPPING and continuous (commaless)

94
Q

how was the genetic code determined?

A

determined through a series of steps
1. overlapping vs non-overlapping, punctuated vs. non-punctuated
2. # of letters in codon
3. # of nt per codon using T4 phage rII insertion/deletion mutations
4. # of nt per codon using synthetic mRNAs
5. ID OF INDIVIDUAL CODONS AND THE AA THEY ENCODE WAS ACHIEVED USING SYNTHETIC mRNAS and tRNA BINDING EXPERIMENTS

95
Q

what are the stop codons?

A

UAG, UGA, UAA

peptide sequence is co-linear with mRNA sequence

these codons are recognized by release factors

96
Q

more than one codon cannot specify the same amino acid (t/f)

A

false

97
Q

what is unique about the genetic code?

A

it is universal

98
Q

what are exceptions to the genetic code?

A

selenocysteine and pyrrolysine are sometimes called the 21st and 22nd AA

encoded by UGA and UAG

99
Q

what factors are needed for translation?

A

mRNA, ribosomes, tRNAs, and protein factors

100
Q

what are the components of the tRNA?

A

contains an acceptor arm (CCA 3’) that carries the AA for the codon
3 step loop structures = a D-arm (dihydroxyl uracil) and a T-arm (ribothymamide), variable loop, and anticodon

Gm is the wobble base

secondary structure = cloverleaf,
3D = it has an L-shape

101
Q

what is the enzyme that attaches AA to tRNAs?

A

aminoacyl-tRNA synthetases (20 total, one for each AA)

they catalyze 2 step reactions involving 3 different molecules –> AA, tRNA, and ATP –> results in charged or aminoacylated tRNA

102
Q

what is the wobble rule of tRNAs?

A

the genetic code is degenerate (multiple codons, or sets of three nucleotides, can code for the same amino acid during protein synthesis)

with exception of SER, ARG, and LEU (6 codons each), this degeneracy always occurs at the 3rd position

103
Q

what is the wobble hypothesis?

A

a single tRNA can decode more than 1 codon because the first base of the anticodon (wobble position which pairs with the 3rd wobble base of the codon) can form additional non-W-C pairs

1st two positions pair according to AU/GC rule

G can pair with C and U
U can pair with G and A

wobble restricted to 3rd codon position (1st anticodon position)
1st and 2nd positive are strictly W-C pairings

104
Q

what is significant about the wobble position?

A

wobble position of anticodon in tRNA is HEAVILY MODIFIED
I = inosine (base found in some tRNAs, derived from A) binds to U,C,A

AGG - UCU instead of UCC
AGU - UCG instead of UCA
UCG - AGU instead of AGC

105
Q

where does translation occur?

A

on the ribosome

106
Q

what are the functions of the subunits of the ribosome?

A

small subunit = decoding functions
large subunit = peptide bond formation functions

bacterial cells only have one type of ribosome in their cytoplasm, eukaryotic cells have two types of ribosomes - one in cytoplasm and others in mitochondria and chloroplasts

107
Q

what are the 4 stages of translation?

A
  • initiation (ribosomal subunits, mRNA, and initiator tRNA assemble together)
  • elongation (ribosome slides along mRNA and synthesizes a polypep)
  • termination (stop codon is reached and polypep is released from ribosome)
  • recycling (recycle translational components, ribosomes split into subunits needed for initiation of new round of translation)
108
Q

what are the 3 binding sties for tRNA on the ribosome?

A

A site (aminoacyl - enter)
P site (peptidyl where the amino acid is added to the polypeptide growing chain)
E site (where the tRNA is released)

anticodon of tRNA interacts with small subunit and the CCA 3’ end interacts with the large subunit

109
Q

explain initiation of translation

A

always begins with binding of SMALL ribosomal subunit to mRNA (30S in bacteria, 40S in eukaryotes)

dedicated INITIATOR tRNA BINDS TO P SITE along with initiation factors

in bacteria, initiator tRNA = charged with formyl-met
in eukaryotes, initiator tRNA = only charged with met
initiator tRNA pairs with AUG

LARGE subunit binds to 70S (bacterial) or 80s (eukaryotic) ribosome

110
Q

how do ribosomes know where to initiate in bacteria?

A

AUG is initiation codon that codes for Met at internal positions
in BACTERIA, there is a ribosome binding site upstream of AUG in mRNA = Shin-Dalgarno mRNA sequence is complementary to 3’ end of 16S rRNA in
mRNA-rRNA base pairing positions 30S over AUG

bacterial specific mechanism

111
Q

how do ribosomes know where to initiate in eukaryotes?

A

using SCANNING mechanism
all eukaryotic mRNAs have CAP at 5’ end
eukaryotic initiation factors bind to 5’ CAP and recruit 40S small subunit

112
Q

what are the steps of elongation?

A

tRNA binding/decoding in A site
peptide bond formation in P site
translocation and tRNA in E site leave s

113
Q

how is the ribosome a ribozyme?

A

2/3 RNA and 1/3 protein
both of its principal functions - decoding and peptide bond formation - are RNA-based activities

it is an RNA molecule with a catalytic function

114
Q

when can translation begin?

A

it can begin before transcription is completed (aka coupling) - DOES NOT OCCUR IN EUKARYOTES
bacteria lack nucleus so transcription and translation occur in the cytoplasm

in eukaryotes, transcription is in the nucleus and translation is in the cytosol

115
Q

what are unregulated genes called?

A

constitutive genes = always on

they proteins that are continuously necessary for the survival of the organism

116
Q

why is gene regulation important?

A
  • metabolism
  • response to environmental stress
  • cell division
117
Q

explain regulation of gene expression in transcription

A

Transcription attenuation is a regulatory mechanism that controls gene expression by causing transcription to end prematurely. It’s a common strategy in bacteria, and is used to maintain a desired level of gene expression and to respond to environmental signals

118
Q

explain regulation of gene expression in translation

A
  • translation repressor proteins - can prevent translation from starting
  • riboswitches - can produce mRNA conformation that influences translation
  • antisense RNA - can bind to mRNA and prevent translation from starting

regulation can occur POST TRANSLATIONALLY

119
Q

what is the function of small effector molecules that increase transcription?

A

they affect transcription regulation
bind to regulatory proteins but NOT to DNA directly

may increase transcription
molecules are INDUCERS because they are INDUCIBLE
bind activators = cause binding to DNA
bind repressors = inhibit binding to DNA

120
Q

what happens when there’s a repressor protein, inducer molecule, and inducible gene?

A

without inducer, repressor will bind to DNA and inhibit transcription

when inducer binds to repressor, DNA transcription can proceed

121
Q

what happens when there’s an activator protein, inducer molecule, and inducible gene?

A

without inducer, activator protein will not bind to DNA and transcription will not take place

with inducer, activator will bind to DNA and transcription occurs

122
Q

what is the function of small effector molecule that inhibit transcription?

A

COREPRESSORS bind to repressors and cause them to bind to DNA
inhibitors bind to activators and prevent them from binding to DNA and transcribing = REPRESSIBLE

123
Q

what happens when there’s a repressor protein, corepressor molecule, and repressible gene?

A

without corepressor, repressor protein will not bind to DNA, transcription occurs

with corepressor, repressor protein will bind to DNA and inhibit transcription

124
Q

what happens when there’s an activator protein, inhibitor molecule, and repressible gene?

A

without inhibitor molecule, activator protein can bind to DNA and transcription occurs

with inhibitor molecule, activator cannot bind to DNA, transcription does not occur

125
Q

what kind of sites do regulators have?

A

DNA binding site and an allosteric site that binds small effector molecule

DNA binding site and allosteric site are coupled

126
Q

what is enzyme adaptation?

A

a particular enzyme appears in the cell only after the cell has been exposed to the enzyme’s substrate

127
Q

what DNA sequences does an operon contain?

A

promoter, operator, structural genes, terminator

128
Q

what are the sequences of the lac operon?

A

LacI repressor = DNA binding protein which can block lacZYA, encoded on separate gene

Lac promoter = DNA site, where RNAP binds, initiates transcription

Lac operator = DNA site, where REPRESSOR binds, between promoter and Z

129
Q

what are the structural genes of the lac operon?

A

lacZ = encodes beta-galactosidase (break down lactose into glucose and galactose, rearranges lactose into allolactose - inducer)
lacY = encodes lactose permease
lacA = encodes beta-galactoside transacetylase

130
Q

what are the two in which the lac operon is regulated?

A
  1. repressor lacI responds to presence/absence of LACTOSE = inducible, negative control mechanism, inducer is allolactose, binds to lac repressor and inactivates it
  2. activator protein - responds to presence/absence of GLUCOSE
131
Q

what does the mutation in lacZY result in?

A

lac- which is unable to use lactose

132
Q

what does lacOc result in?

A

unable to bind the repressor so the lac operon is ALWAYS ON regardless if lactose is present or absent

it is CIS-ACTING (only affects expression of adjacent, downstream lac operon)

ex: WT lacZ cis to Oc = always on (in Oc/O+)
WT lacY in cis to O+, lacY on only when lactose is present

133
Q

what does lacI result in?

A

lacI- is always on
lacI- is recessive to lacI+ (I+ is inducible)
one copy of lacI+ encodes enough protein to regulate 2 operons (I+ is TRANS ACTING, can regulate Z+ and Y+ on different DNAs)

134
Q

what does lacIS result in?

A

it is a SUPER-REPRESSOR
represses even in the presence of inducer
lacIS is DOMINANT to lacI+

135
Q

explain inducible and repressible regulation of operons involved in catabolism and anabolism

A

operons involved in catabolism are INDUCIBLE

operons involved in anabolism are REPRESSIBLE

136
Q

what are translational regulatory proteins?

A

recognizes sequences within mRNA and regulate translation of mRNA

these proteins act to inhibit translation aka translation repressors

they inhibit translation in 2 ways:
1. binding next to the Shine-Dalgarno sequence and/or start codon (sterically hinder ribosome from initiating translation)
2. binding outside the Shine-Dalgarno/start codon region (stabilize an mRNA secondary structure that prevents initiation)

another way to regulator is via synthesis of antisense RNA

137
Q

what is the funciton of proteim ompF?

A

important in osmoregulation
produced at LOW osmolarity, at HIGH osmolarity its synthesis is DECREASED

micF inhibits ompF at high osmolarity but does NOT code for a protein

micF is an antisense RNA (complementary) of ompF

137
Q

what are some posttranslational regulation mechanisms?

A

feedback inhibition
phosphorylation
acetylation
methylation

138
Q

what is the function of riboswitches?

A

it can regulate transcription of translation, or splicing

RNA exists in 2 different secondary conformations - one that allows gene expression and another that inhibits it
conversion between forms is due to binding of a small molecule, which is a product of the pathway being regulated

139
Q

explain riboswitch of TPP

A

genes for TPP synthesis are found in the thi operon which is regulated by a riboswitch

riboswitch will determine if it continues or not (thiMD mRNA is ALWAYS made)
if TPP is low = transcription continues and mRNA is made

if TPP is high = terminator stem-loop forms and transcription is attenuated/terminated at U-rich sequence

140
Q

what do the regulatory transcription factors influence?

A

influence ability of RNA pol II to being transcription of a PARTICULAR gene

141
Q

what is combinatorial control?

A

every promoter’s activity is influenced by multiple transcription factors

common concepts contributing to combinatorial control
- one or more activator proteins may stimulate transcription
- one or more repressor proteins may inhibit transcription
- activators and repressors may be modulated by: binding of small effector molecules, protein-protein interactions, and covalent mods.
- regulatory proteins may alter nucleosomes near promoter
- DNA methylation may inhibit transcription by: preventing binding of activator protein and recruiting proteins that compact chromatin

142
Q

what is important about enhancers/silencers?

A

they are orientation-independent
can function in forward or reverse orientation
most are located within a few hundred nt upstream of the promoter

143
Q

how do regulatory transcription factors influence transcription by pol II in eukaryotes?

A

they DO NOT bind directly to RNA pol
repressors don’t block pol II binding and activators don’t recruit pol II directly

3 ways that communicate effects of regulatory transcription factors to the transcription machinery are:
- regulation via TFIID either directly or through coactivators/corepressors
- regulation via mediator
- regulation via alternations in chromatin conformation

144
Q

what are the functions of coactivators?

A

proteins that increase transcription, but DO NOT BIND TO DNA DIRECTLY

145
Q

what is the function of the mediator?

A

aid in phosphorylation of CTD of pol II, happens at the end of initiation, allowing it to enter elongation

phosphorylation allows pol II to move away from the promoter region and transcribe downstream DNA

repressor prevents phosphorylation, pol II is trapped at promoter and CANNOT proceed to elongation phase

146
Q

when can the transcription factor bind to DNA?

A

it can only bind when a small molecule is bound to it
active form of TF is homo/hetero dimer

147
Q

describe action of glucocorticoid hormones

A

hormone diffuses into cell, binds to a glucocorticoid receptor –> triggers release of Hsp90 –> NLS is now exposed –> 2 receptors dimerize and travel to nucleus –> GRE are DNA sequences that function as enhancers (located near dozens of different genes, so hormone can activate many genes

148
Q

describe the activity of the CREB protein

A

cAMP acts as a second messenger that activates PKA –> phosphorylated CREB binds to DNA and stimulates transcription (CREB protein recognizes response element with consensus sequence 5’ TGACGTCA 3’) –> unphosphorylated CREB can bind to DNA but cannot activate transcription –> activation happens when CREB is bound by coactivator (CBP) which contacts the transcription machinery to activate it

149
Q

what are the conformations of the chromatin?

A

closed = chromatin is VERY TIGHTLY packed, transcription may be difficult or impossible

open = chromatin is accessible to transcription factors, transcription takes place

150
Q

what are the 3 types of changes to chromatin that affect accessibility of DNA?

A
  1. chromatin remodeling - moving nucleosomes on chromatin
  2. chemical modification of chromatin
  3. swapping common histones for variants
151
Q

what are the 3 ways in which chromatin remodeling complexes change chromatin structure?

A
  • change in position of nucleosomes (spacing to be more opened)
  • eviction of histone octamers (histone removed leaving only DNA)
  • change in composition of nucleosomes
152
Q

what are the 5 histone genes?

A

H1, H2A, H2B, H3, H4

153
Q

what is the histone code?

A

pattern of recognition mechanism

DNA transcription is largely reculated by post-translational mod to histone proteins

pattern of histone mods provide binding sites for proteins like TFs

these proteins bind based on histone code and affect transcription

154
Q

what is acetylation associated with?

A

increased transcription of DNA
hypoacetylation = decreased transcription

acetylated lyse binds DNA less well, creates a more opened complex = increase transcription

155
Q

why is the ChIP-sequence important?

A

it maps locations of specific nucleosomes within a genome

allows determination of:
- where nucleosomes are LOCATED
- where histone variants are FOUND
- where covalent mods of histones OCCUR

156
Q

what is methylation associated with?

A

carried out by DNA methyltransferase
common in SOME EUKARYOTIC SPECIES, but not all
DNA methylation INHIBITS eukaryotic gene transcription

methylation of cytosine in CpG di-nt

ASSOCIATED WITH INACTIVE GENES (INHIBIT TRANSCRIPTION) and leads to gene silencing
methylation may influence binding of TFs

157
Q

explain CpG islands

A

many genes contain CpG islands near their promoters

in housekeeping genes (expressed all the time) –> CpG islands are unmethylated, housekeeping genes are also expressed in most cell types

Diana's Flashcards (8 decks)

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