Lecture 6 - RNA Synthesis

Question 1

Human genome (2)

Answer 1

3.2 x 109 nucleotides.

23 chromosomes - 22 autosome and 1 sex pair.

Question 2

Homologous chromosomes

Answer 2

Chromosome pairs (one from each parent) that are similar in length, gene position, and centromere location. The position of the genes on each homologous chromosome is the same, however, the genes may contain different alleles.

Question 3

Centromeres (3)

Answer 3

Primary constriction - looks nipped in at the chromosome under the microscope.
Keeps chromosomes attached to the mitotic spindle during mitosis so new daughter cells have a copy.

Question 4

Telomeres (3)

Answer 4

Located at the end of the chromosomes.
Very relative DNA, 6 base pair repeats, protects the end of the chromosome - so the genes carried aren’t destroyed.
Prevents strand linkages so that chromosomes don’t join together.

Question 5

Gene (3)

Answer 5

Unit of heredity; contains instructions for an organism’s phenotype.
DNA segment containing instructions for making a particular product, including the regulatory elements.
Contains a lot of regulatory regions.

Question 6

Transcription (2)

Answer 6

Synthesis of mRNA transcript from DNA.

Same language – nucleic acid to protein.

Question 7

Translation (2)

Answer 7

Protein production from mRNA transcript.

Different languages – nucleic acid to protein.

Question 8

Human RNA Polymerase (3) (1)

Answer 8

I- Most ribosomal RNA (rRNA).
II - Protein coding, microRNA (miRNA), non-coding RNA.
III - Transfer RNA (tRNA), 5S rRN, other small RNAs.

Reads in 3’ to 5’ direction to produce pre-mRNA from 5’ to 3’ direction.

Question 9

RNA synthesis in humans (3)

Answer 9

More than one RNA polymerase can work on one gene.
Many transcripts from a gene simultaneously.
Relatively quick 1.25-1.74kb per min.

Question 10

Transcription factors (3)

Answer 10

Proteins required to initiate or regulate transcription in eukaryotes.
Assemble on promoter to position on RNA Polymerase II.
Pull apart DNA helix and expose template strand.

Question 11

RNA vs DNA (4)

Answer 11

Ribose nucleotides not deoxyribose nucleotides.
Uracil instead of Thymine.
Single stranded not double.
Shorter than DNA.

Question 12

mRNA (3)

Answer 12

Codes for a particular protein from the cell nucleus to the cytoplasm where proteins are synthesised.
Long single stranded molecule of upto thousands of nucleotides which can contain one gene only.
Makes up less than 5% of the total cellular RNA.

Question 13

tRNA (5)

Answer 13

80 nucleotides long.
Single stranded.
Makes up 10-15% of the cells RNA.
64 different types of tRNA with a different anticodon complementary to specific codons.
AA are attached to tRNA by specific enzyme aminoacyl tRNA synthase - so each tRNA adaptor is paired with the correct anticodon.

Question 14

How does RNA polymerase know where to start transcribing? (1)

Answer 14

Promotor sites which are specific sequences of nucleotides which binds with RNA polymerase.

Question 15

Gene-Specific Regulation of Transcription

Answer 15

Additional upstream sequences are needed for gene-specific regulation of transcription (e.g. enhancers, proximal control elements).

Question 16

What is a consensus sequence? (4)

Answer 16

A sequence of DNA having similar structure and function in different organisms.
Amalgamation of several promoter regions into one that is the same in many organisms.
Prokaryotes - TATAAT - Pribnow box.
Eukaryotes - TATA - Hogness box.

Question 17

TATA box (4)

Answer 17

Promoter region in eukaryotes.
A TATA box is a DNA sequence that indicates where a genetic sequence can be read and decoded. It is a type of promoter sequence, which specifies to other molecules where transcription begins.
Has a consensus sequence of TATAAA it is found 25 nucleotides to the start of where the gene is found.

Question 18

CAAT box (1)

Answer 18

Consensus sequence of GGNCAATCT it is found 75 nucleotides to that start of where the gene is found (upstream of the gene).

Question 19

Enhancer sequences (4)

Answer 19

Not found in prokaryotic cells.
A sequence on which a protein binds and it forms a complex which promotes the process of transcription and increases effciency of RNA Polymerase.
Found up/downstream.
Usually 1000 nucleotides away from the gene that is being transcribed.

Question 20

Requirements of transcription (4)

Answer 20

rNTPs - Building blocks of RNA.
Promoter regions - “Bookmark” so RNA Polymerase knows where to transcribe.
Transcription factors - used for requirement of RNA Polymerase II in eukaryotes. As it does not recognise or bind to promoter regions unlike prokaryotes so a TF is needed.
Enhancers and silencers - Elements which can enhance or supress he transcription of a gene.

Question 21

Kozak consensus sequence (3)

Answer 21

Occurs on eukaryotic mRNA.
Has the consensus (gcc)gccRccAUGG.
Plays a major role in the initiation of the translation process.
25 nucleotides upstream from AUG start codon.

Question 22

UTRs (Untranslated regions) (3)

Answer 22

UTRs are transcribed but not translated.
5’ UTR: regulation of translation.
3’ UTR: mRNA stability & miRNA binding.

Question 23

Initiation (9)

Answer 23

TFIID [made up of multiple subunits including TBP (TATA binding protein)] binds to promoter region.
TFIIA and TFIIB bind and TFIIA stabilises the complex. TFIID-TFIIA-TFIIB.
TFIIF associates with RNA Polymerase II before it binds to any other TF. TFIIF stabilises the RNA Polymerase II while it’s contacting TBP (TFIID) and TFIIB.
Then initiation is completed when TFIIE and TFIIH bind, then RNA Polymerase II assembles at the promoter. Forming the transcription initiation complex.
Forming a huge complex TFIID-TFIIA-TFIIB-TFIIF-TFIIE-TFIIH.
TFIIH unwinds/pulls apart the DNA helix called a cistron around the initiation site and phosphorylates RNA Polymerase II.
Phosphorylated RNA Polymerase II is released from the complex and begins transcription.
ATP is needed to form RNAs transcript.

Question 24

Cistron (1)

Answer 24

A section of a DNA or RNA molecule that codes for a specific polypeptide in protein synthesis.

Question 25

Elongation (4)

Answer 25

After 25 nucleotides of RNA are synthesised the 5' end is modified. Methyl transferase methylates a guanine at position 7 giving rise to 7 methylguanine cap. Cap protects the 5' end from activity of 5' exonucleases (which digest RNA). 5' acts as a 'check' if the ribosome does not detect the 5' cap it will not start, it will not leave the cell (it will be degraded). RNA Polymerase II continues along the DNA making pre-mRNA until it reaches the termination sequence.

Question 26

Termination (8)

Answer 26

Near the stop codon there is a polyadenylation/termination sequence, AAUAAA, after the stop codon. Specific endonuclease (CPSF - cleavage and polyadenylation specificity factor)recognises the termination signal and cleaves after the polyadenylation/termination signal. Polyadenylate polymerase adds 250 adenosine monophosphates to the 3' end of the pre-mRNA called the poly A tail. Longer = More stable. Poly A tail protects it from degradation and promotes the nuclear transport of mRNA. Protects it from RNAses which digest the RNA in the cytoplasm which moves out of the nucleus. If there is no guanine cap or poly A tail there will be no translation.

Question 27

How is the primary transcript is processed? (3)

Answer 27

Capping. Polyadenylation. Splicing.

Question 28

Capping (5)

Answer 28

5’ end modification. Guanine nucleotide: 1. 5’-5’ triphosphate bridge 2. Methylated at 7 position. Capping enzyme complex. Co-transcriptional modification.

Question 29

Polyadenylation (3)

Answer 29

``` Cleavage signal (AAUAAA). Cleavage by specific endonuclease. Addition of tail by poly (a) polymerase. ```

Question 30

Introns (3)

Answer 30

Non-coding sequences within genes. Size: <100 - >700,000 nucleotides Median: 1800 nucleotides (exon: 123 nucleotides). Number/gene: 0 (e.g. histones) – 78 (dystrophin) Median: 7-9.

Question 31

Post transcriptional modification - Splicing RNA

Answer 31

Initial mRNA transcribed = pre-mRNA = exact copy of DNA = contains introns/exons.

Question 32

Spliceosomes (6)

Answer 32

A spliceosome is a large and complex molecular machine found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from snRNAs and approximately 80 proteins. The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript. Each spliceosome is made up of 5 small nuclear RNAs and a range of associated protein factors, they make RNA-protein complexes called snRPS (small nuclear ribonucleoproteins). The snRNAs that make up the major spliceosome are U1, U2, U3, U4, U5, U6, which are rich in uridine. snRNAs are transcribed by RNA Polymerase II/III.

Question 33

Splicing RNA process

Answer 33

From the 5’ end they are GU, the A branch site, a pyrimidine rich region and the 3’ region and the 3’ AG, the GU and AG sequences define the beginning and end of the intron. The intron is cleaved at the 5’ GU sequence and forms a lariat (looped structure which does join with the branch site) at the A branch site. The bonding of the guanine and adenine takes place via transesterification in which a hydroxyl group on the carbon atom of the adenine ‘attacks’ the bond of the guanine nucleotide at the splice site (ss). The guanine residue is cleaved from the mRNA and joins onto the adenine forming a looped structure. The 3’ is cleaved at the AG point and the two exons are lighted together.

Question 34

Alternative splicing / Differential splicing (6)

Answer 34

Exon Skipping 95% of genes. A regulatory process that results in a single gene coding for multiple proteins, in this process exons are selectively spliced depending on which protein is to be formed. Different arrangement of exons produces different proteins as a different combination of exons is ligated together (ligase). Multiple types of mRNA made from same gene = Large variety of proteins.

Question 35

Examples of alternative splicing (4)

Answer 35

Troponin, pyruvate kinase. Isoforms of glucocorticoid receptor. Tropomyosin. Myosin.

Question 36

Exporting from the nucleus (4)

Answer 36

CBC = Cap-binding complex. TREX = Transcription-coupled export complex. EJC = Exon-junction complex. Nuclear-pore complex.