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DNA and RNA synthesis Flashcards

1
Q

What is personalised medicine?

A
  • Move away from ‘one size fits all’ approach to treatment and care of pxs with particular condition
  • we are all unique, health being determined by differences, lifestyles & environment
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2
Q

100,000 Genomes Project

A

100,000 Genomes Project aims to bring benefits of personalised medicine to NHS
-Aim to complete by end of 2017 from 70,000 pxs
By combining and analysing info about genome, patterns identified to help determine individual risk of developing disease; detect illness earlier; determine most effective interventions to help improve health
-e.g. medicines, lifestyle choices, simple changes in diet

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3
Q

Protein synthesis

A

DNA –> transcription –> mRNA –> translation –> protein
Transcriptoin in nucleus
Translation in cytoplasm

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4
Q

Genome is fixed, cells are dynamic

A

Genome: static, every cell in our body has copy of same genome
Cell: dynamic, responds to external conditions.
-most cells follow cell cycle of division
-cells differentiate during development

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5
Q

How much of genome is translated into proteins?

A

3%

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6
Q

What are pseudogenes?

A

Section of chromosome that is an imperfect copy of functional gene
-Related to real genes and contain biological and evolutionary histories within their sequences

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7
Q

How much of genome is transcribed but not translated?

A

25% of the genome

Not associated with protein-coding genes

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8
Q

How many non-functional pseudogenes in the genome?

A

~20,000

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9
Q

ENCODE project

A

Encyclopaedia of DNA Elements project

  • systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification
  • assigned biochemical functions for 80% of genome
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10
Q

Differences between mRNA and DNA

A

mRNA is shorter than DNA (contains info to make one single polypeptide chain i.e. one protein)
DNA = 2 strands in double helix, mRNA = 1 strand
DNA = adenine, guanine, cytosine, thymine
RNA = adenine, guanine, cytosine, uracil
Ribose (RNA): OH gp on 2’ C
Deoxyribose (DNA): H hp on 2’ C

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11
Q

How do thymine and uracil bind to adenine?

A

Via H-bonds

Very similar

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12
Q

What is transcription?

A

Process whereby information in a gene in a DNA strand is transferred to an RNA molecule

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13
Q

Coding and template strand

A

Genetic info carried on coding strand, other one template.

-the strand that serves as coding template for one gene may be non-coding for other genes in same chromosome

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14
Q

mRNA sequence complimentary/ identical to which DNA strand

A

Complimentary to template strand

Identical to coding strand (except from T-U)

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15
Q

How to find start of gene on coding strand of DNA

A

RNA polymerase binds to promoter sequences

-one or more short sequences upstream of start of each gene i.e. slightly closer to 5’ end

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16
Q

RNA polymerase

A
  • synthesis of RNA requires DNA-dependent RNA polymerase
  • does not require primer but does need DNA template
  • 3 found in eukaryotes : I, II and III
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17
Q

RNA polymerase I

A

Nucleolar region of nucleus, transcribes large ribosomal RNA

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18
Q

RNA polymerase II (view pic on Word)

A

mRNA precursors

  • composed of several subunits
  • requires several accessory proteins (transcription factors)
  • all added to complex in defined order to initiate & carry out transcription
  • TFIID recognises TATA box and ensures correct site is used
  • enzyme wraps around both strands and is big enough to enclose promoter and beginning of gene
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19
Q

RNA polymerase III

A

Small RNAs (tRNA), 5S ribosomal RNA and other small DNA sequences

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20
Q

What do most RNA polymerase II genes have?

A

TATA box 25 - 35 bases upstream of initiation site

-affect transcription rate and determines location of start site

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21
Q

Promoters

A

Basal promoter contains TATA box and found in all protein-coding genes

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22
Q

What differs from gene to gene

A

Structure and associated binding factors

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23
Q

Enhancers

A

DNA sequences which can control efficiency and rate of transcription
-regulate expression of genes in specific cell type and control timing of gene expression (effects can be powerful)

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24
Q

What type of elements are promoters and enhancers?

A

‘Cis’ acting elements

-on same molecule of DNA as the gene they regulate

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25
Strong or weak promoters and enhancers
According to effects on transcription rates and thus gene expression -changes in promoter strength, deleterious effects on cell--> disease
26
Tumour promoting viruses and promoters
Transform healthy cells by inserting strong promoters near growth-stimulating genes -translocations in some cancer cells place genes that should be 'turned off' in proximity of strong promoters and enhancers
27
Where is promoter necessary to start transcription located?
Usually on 5' side of gene to be transcribed
28
Where can enhancers affect transcription from?
From afar 5' or 3' transcription start site In introns or exons Non-coding strand
29
Are enhancers and promoters always close together?
Can be 1000s of nucleotides away from promoters, brought into close proximity by looping of DNA -looping due to interactions between proteins bound to enhancers and those bound to promoter
30
Protein facilitating/ ihibiting looping
Facilitating: activators Inhibiting: repressors
31
Enhancers and TF
Enhancers contain binding site sequences for transcription factors and enhance/ upregulate transcription -active enhancers bound by activating TF and brought into proximity of target promoters by looping
32
Basal transcription factors
TF bind to promoter and enhancer sequences and recruit RNA polymerase. Basal TF required at every promoter site for RNA polymerase interaction (TFIID)
33
Are TF cis or trans
TFs are 'trans' acting factors as encoded by different gene to that being regulated
34
What happens once polymerase is attached
Unwinds double helix over short length and splits them apart | -bubble of about 10 bases
35
What bond does RNA polymerase catalyse?
Sugar-phosphate bond between 3' -OH of ribose and 5' PO4
36
What does order of bases in DNA template strand determine?
Order of bases in transcript
37
Where are nucleotides added to?
The 3' -OH of growing chain
38
What happens when polymerase moves down DNA?
DNA zips back up - bubbles moves along chain and growing RNA tail detaches from template
39
How is RNA a 3D structure if it is only single stranded?
Base-pair interactions between complementary sequences found elsewhere on same molecules allow RNA molecule to fold. Determined by sequence of nucleotides -folding similar to way polypeptide chain folds in protein
40
What functions does folding ability of RNA give?
Some RNA molecules have structural and catalytic functions
41
Termination of pol I genes
By a termination factor | Leads to hair pin loop which causes RNA polymerase to pause and release transcript
42
Termination sequence pol III genes
Includes a polyuracil stretch | Causes RNA pol to pause and release transcript
43
Transcription of pol II genes
Can continue for 100s or 1000s of nucleotides beyond end of coding sequence
44
Mature pol II mRNAs
Polyadenylated at 3' end = poly(A) tail | AAAAAAAAAAAA
45
Polyadenylation and termination
Same concensus sequence Sequence AAUAAA found near polyadenylation site of eukaryotic mRNAs -required for accurate and efficient cleavage and polyadenylation of premRNAs in vivo
46
DNA to mRNA as RNA pol moves down gene
1. CAP on 5' end to stabilise mRNA (essential for transport of RNA out of nucleus) 2. Alternative splicing 3. Poly(A) tail on 3' end - cleavage at AAUAAA (stop codon) by endonuclease then multiple adenosine (up to 250) added - needed for release of polymerase from DNA template
47
Capping pre-mRNA
Takes place during transition from transcription initiation to elongation, when pre-mRNA only 20-40 nucleotides long - protects from degradation - serves as assembly point for proteins needed to recruit small subunit of ribosome to begin translation
48
Alternative splicing
Several variants of same protein produced by one gene | Mutations at splice sites can result in aberrant or truncated proteins that do not function properly
49
Alternative splicing - transciptome and proteome diversity
- alternative selection of splice sites within a pre-mRNA - leads to production of different mRNA isoforms of gene - alters compositiong and function of encoded protein - plasticity allows for disease development - cancer
50
What is a spliceosome and how is it formed
Exons defined by short, degenerate classical splice site sequences at intron/ exon borders Components of basal splicing machinery bind to classical splice-site sequences and promote assebly of multi-component splicing complex - spliceosome
51
Spliceosome function
1. Recognition of intron/ exon boundaries | 2. Catalysis of cut and paste reactions which remove non-coding introns and stitch flanking exons back together
52
Spliceosome types
Two types - major: removes 99.5% of introns - minor: removes remaining 0.5%
53
What do spliceosomes contain
Proteins (over 300 different proteins associated with human spliceosome) and RNAs
54
Spliceosome and disease
- mutations altering splice site or spliceosome proteins - mis-splicing = rapid degeneration of mRNA - mis-regulation of spicing factor levels = cancer
55
Types of RNA
1. mRNA 2. rRNA 3. tRNA 4. Non-coding RNA (ncRNA) 5. Small nuclear RNA (snRNA) 6. Small nucleolar RNA (snoRNA) 7. Micro RNA (miRNA)
56
mRNA specificity
Specific for only certain types of cells, encoding proteins needed by 'specialised' cells e.g. mRNA for haemoglobin in precursors of RBCs
57
mRNA size and variety
Wide range of sizes reflecting size of polypeptide | Many common to most cells, 'housekeeping' proteins needed by all cells e.g. enzymes of glycolysis
58
Types of rRNA in eukaryotes
4 kinds 18S - 1 of these along with other proteins make the small subunit of the ribosome 28S, 5.8S & 5S - one each of these, along with 45 other proteins used to make the large subunit of the ribosome
59
What is rRNA?
Builds ribosomes, the machinery for synthesising proteins by translating mRNA
60
What does S stand for in types of rRNA?
Svedberg unit | Sedimentation rate, related to mass and shape
61
How many types of tRNA in a typical eukaryotic cell?
32 | Each kind carries, at 3' end, one of 20 amino acids i.e. most amino acids have more than one tRNA
62
What is non-coding RNA
Around 2% of genome corresponds to protein-coding sequences remainder - 'junk' sequence -previously pathologic alterations due to aberration of gene sequence or altered promoter activity
63
How much of genome is actively transcribed?
90%, this part is more complex
64
What can ncRNA do
Can modify protein levels by mechanisms independent of transcription Plays major roles in cellular physiology, development, metabolism and is implicated in disease process
65
What is snRNA
Small nuclear RNA Transcription of genes for mRNA, rRNA and tRNA produces large precursor molecules or primary transcripts Must be processed in nucleus to allow export to cytosol of functional molecules Some processing steps mediated by snRNAs Several snRNAs are part of spilceosome
66
snoRNA
Small nucleolar RNA Participate in making ribosomes by helping cut large precursor of 28S, 18S and 5.8S Modify many nucleotides in rRNA, tRNA and snRNA e.g. can add methyl groups to ribose Implicated in alternative splicing of pre-mRNA Template for synthesis of telomeres
67
How are snoRNAs made in vertebrates?
Made from introns removed during RNA processing
68
miRNA
Micro RNA Tiny RNA molecules regulate gene function post-transcriptionally Very small, 18-25 nucleotides Bind to mRNA and cause degradation - inhibits protein synthesis Regulation of developmentally timed events Exhibit tissue-specific and/ or developmental stage-specific expression Cancer & tooth development
69
How many protein-coding genes are under control of miRNAs?
More than 1/3

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