L28,29: Gene Expression I: Transcription and RNA Processing

Question 1

DNA Packing

Answer 1

• DNA in double helix form is tightly wound around histone proteins, forming nucleosomes (DNA -ve,
  histone protein +ve therefore bind tightly)
• length of linker DNAis constant within cells but can vary between species.
• The combination of proteins + DNA is called “chromatin”
• Interphase: length of nucleosomes coil to form thicker chromatin (30nm) due to linker DNA and nucleosome interactions.
• thicker chromatin fibre folds along non-histone protein scaffold forming looped domains, (fibres now 300nm thick).
• further folding of looped domains is seen during metaphase

metaphase chromosome consists of 2 chromatids (each 700nm in width).

Mitosis: Pairs of chromosomes segregate properly into the daughter cells.

Question 2

Bacteria Transcription

Answer 2

It is the synthesis of mRNA from a section of DNA and takes place in the CYTOPLASM!

Initiation: 
RNA polymerase (doesn't need primers) binds to the promotor,  this is a sequence of DNA where transcription is initiated, RNA polymerase moves downstream of DNA sequence and causes  hydrogen bonds between the bases to break and bases on the DNA strand are exposed, hence the DNA double helix is being "unzipped".

Elongation:
As the RNA Polymerase moves downstream, unzipping the DNA act elongates the RNA transcript 5’ -> 3’. In the wake of transcription DNA strands reform a double helix

Termination: Different for bacteria and eukaryotes

Termination:
An RNA sequence is transcribed and acts as a terminator, this causes RNA polymerase to detach from the DNA and for the mRNA transcript to be released, this mRNA transcript can be translated without further modification.

Question 3

What is mRNA

Answer 3

Messenger RNA:

• mRNA is responsible for formation and maintenance of different cell and tissue types
• mRNA transcribes the DNA code. Carries a copy of DNA code from the nucleus to the ribosome. (Production of mRNA is essential since DNA itself is too large to pass through the nuclear membrane.)

Question 4

What is tRNA

Answer 4

Transfer RNA: translates the DNA code. Each tRNA molecule has a site, at the 3’ end, to carry a specific amino acid from the cytoplasm to the ribosome, and on the other end of the molecule it has an anticodon; this anticodon base-pairs with a complementary codon on mRNA,

Anticodons are written 3’ -> 5’ this allows anticodons to aline with codons which are written 5’->3

Question 5

rRNA

Answer 5

Ribosomal RNA: is the main component of ribosomes, along with protein.

Question 6

snRNA

Answer 6

Small Nuclear RNA

Question 7

Describe RNA

Answer 7

• Single stranded
• nucleotide (ribose sugar, phosphate and base)
• Bases: adenine, uracil, cytosine and guanine

Question 8

Describe DNA

Answer 8

• Double helix, composed of two anti-parallel strands
• nucleotide (deoxyribose sugar, phosphate and base)
• Bases: adenine, thymine, cytosine and guanine

Question 9

RNA Splicing

Answer 9

RNA Splicing forms a mature mRNA transcript by removing introns from the mRNA primary transcript and joining the exons together (the order of exons is unchanged); this produces an mRNA molecule with a continuous coding sequence

Question 10

Introns

Answer 10

Regions of DNA which don’t code for proteins

Question 11

Exons

Answer 11

Regions of DNA which code for proteins

Question 12

Protein synthesis

Answer 12

the process by which proteins are made. Involves the transcription and translation of DNA sequences. Occurs in the cytoplasm.

Question 13

Genotype

Answer 13

The genetic constitution of a cell’s DNA sequence

Question 14

Phenotype

Answer 14

Both the cell’s physical and chemical state. It is determined by the proteins produced as a result of gene expression.

Question 15

Alleles

Answer 15

Different forms of a gene which code for the same protein.

Question 16

Requirements of mRNA

Answer 16

• The messenger should be a polynucleotide
• The base composition should reflect that of the DNA
  that specifies it
• The messenger should be very heterogeneous in size because genes vary in length
• It should be synthesised and degraded very rapidly

Question 17

Explain why tRNA is not a suitable messenger

Answer 17

-Too homogenous, too small.

Question 18

Role in Transcription: Template strand

Answer 18

• Template strand (“Anti-sense strand”): Provides a template to be copied, allows for ordering the of sequence of nucleotides in the RNA transcript.

Question 19

Role in Transcription: Non-template strand

Answer 19

-Non-template strand (“Sense strand”), Nucleotide are identical to the condos except T is present In DNA instead of U which is present in RNA.

Question 20

Role inTranslation Codon

Answer 20

-A codon is a triplet of nucleotides and it specifies the amino acid to placed at the corresponding position along the polypeptide.

Question 21

Role in Translation: Stop codon

Answer 21

Stop codons signal the end of translation (there are 3)

Question 22

Role in Transcription: Eukaryotic RNA Polymerases

Answer 22

• RNA polymerase I (PolI): rRNA
• RNA PolII: mRNA and some snRNAs
• RNA PolIII: tRNA, 5S rRNA and 7S RNA of the signal recognition particle

Question 23

Describe the molecular Components of Transcription

Answer 23

• RNA synthesis is catalyzed by RNA polymerases, which separate the DNA strands and join together the RNA nucleotides.
• The RNA is complementary to the DNA template strand
• RNA polymerases (in contrast to DNA PolIII) do not need primers
  • RNA synthesis follows the same base-pairing rules as DNA, except that uracil substitutes for thymine

Question 24

Eukaryotic Transcription

Answer 24

It is the synthesis of mRNA from a section of DNA and takes place in the NUCLEUS!

Initiation:

Transcription factors are proteins which bind to the eukaryotic promotor, this is a sequence of DNA where transcription is initiated and for eukaryotes includes a short sequence called the “TATA box”, after the attachment of transcription factors it is then possible for RNA polymerase II to also bind to the promotor, this causes hydrogen bonds between the bases to break and bases on the DNA strand are exposed, hence the DNA double helix is “unzipped”.

The complete assembly of transcription factors and RNA Polymerase II is called “the transcription initiation complex” this allows for transcription to be initiated.

Elongation:
RNA Polymerase moves downstream, unzipping the DNA and elongating the RNA transcript by adding nucleotides to the 3’ end of the growing RNA molecule. In the wake of transcription the new RNA molecule peels away from the DNA template strand and the DNA strands reform a double helix.

Termination:
RNA Polymerase II transcribes a sequence of RNA called the “polyadenylation signal sequence” in the pre-mRNA.
The RNA nucleotides produced by transcription are immediately bound by proteins in the nucleus; downstream of these RNA nucleotides proteins cut the pre-mRNA transcript free from the RNA Polymerase II, causing the pre-mRNA to also be released, it must then be processed by splicing.

pre-mRNA Splicing:

The spliceosome is a large complex made of proteins and small RNAs. The spliceosome binds to short sequences of nucleotides along an intron, this causes the intron to be released rapidly and the spliceosome then joins the two exons which were previously flanked the intron. small RNAs also serve as a catalyst for this process.

During splicing each end of a pre-mRNA molecule is modified in a particular way
– The 5′ end receives a modified nucleotide 5′ cap
– The 3′ end gets a poly-A tail

These modifications share several functions
– They seem to facilitate the export of mRNA to the cytoplasm
– They protect mRNA from hydrolytic enzymes
– They help ribosomes attach to the 5′ end

Question 25

Give three properties of RNA enable it to function as an enzyme

Answer 25

– It can form a three-dimensional structure because of its ability to base-pair with itself

– Some bases in RNA contain functional groups that may participate in catalysis

– RNA may hydrogen-bond with other nucleic acid molecules

– snRNAs are examples of catalytic RNAs – Ribozymes (RNA enzymes) are also catalytic RNAs

Question 26

Provide evidence for mRNA Splicing in eukaryotes

Answer 26

• FAR too much DNA in animal cells for the anticipated number of genes.
• Long RNAs in the nucleus compared with short mRNAs in the cytoplasm.

Question 27

Describe self-Splicing RNA Introns

Answer 27

The intron RNA acts as a ribozyme and catalyses its own excision

small RNAs within the spliceosome base-pair with nucleotides at specific sites along the intron, small spliceosome RNAs then catalyse cutting of the pre-mRNA and the splicing together of exons, during this process the intron is released for rapid degradation.

Question 28

Explain how one gene can code for many proteins?

Answer 28

Alternative splicing of a primary mRNA transcript is the process by which exons are included or excluded from a mature mRNA transcript. As a result the same primary transcript can be used to produce several mRNA molecules each with different base sequences coding for different polypeptides.

*Number of different proteins an organism can produce is greater than its number of genes

Question 29

Describe Translation

Answer 29

Initiation:

• In order for translation to take place first, a small ribosomal subunit binds with mRNA and a special initiator tRNA. The small subunit moves along the mRNA until it reaches the the start codon AUG, this signal the start of translation. Proteins called initiation factors bring in the large subunit that completes the translation initiation complex

Elongation:
• tRNA molecules transport amino acids from the cytoplasm to the ribosome. These amino acids are added one by one to the C-terminus of the growing chain and proceeds along the mRNA in a 5′ → 3′ direction, (The ribosome and mRNA move relative to each other, codon by codon);each of these additions involve proteins called elongation factors

• Elongation occurs in three steps:

1) codon recognition
2) peptide bond formation
3) translocation

*For (1&3) Energy expenditure occurs

Termination of Translation

• Elongation continues until a stop codon in the mRNA reaches the A site of the ribosome
• The A site accepts a protein called a release factor
• The release factor causes the addition of a water molecule instead of an amino acid
• This reaction releases the polypeptide, and the translation assembly comes apart

Question 30

Describe, for both eukaryotes and bacteria, how the accuracy of translation is ensured

Answer 30

In both bacteria and eukaryotic cells tRNA molecules transport amino acids from the cytoplasm to the ribosome. This process is regulated by t Aminoacyl-tRNA synthetases, there are 20 different types of this enzyme and they ensure the correct paring of amino acid with tRNA molecules.

Question 31

Describe the structure of a ribosome:

Answer 31

A ribosome has three binding sites for tRNA
– The P site holds the tRNA that carries the growing polypeptide chain
– The A site holds the tRNA that carries the next amino acid to be added to the chain
– The E site is the exit site, where discharged tRNAs leave the ribosome

Question 32

Definition: Transcription

Answer 32

the synthesis of mRNA using information in DNA

Question 33

Definition: Translation

Answer 33

the synthesis of a polypeptide, using information in the mRNA, takes place at the ribosome.