PART II: GENES & GENOMES: SEQUENCE VARIATION AND ANALYSIS

Question 1

What are examples of regulatory sequences like promoters and RBS respectively?

Answer 1

• TTGACA-17bp-TATAAT

- GGAGG near the start codon of a gene encoding a protein

Question 2

What two things does gene annotation encompass?

Answer 2

1. Predicting and MARKING the position of genes and OTHER elements on a genome sequence
2. Predicting protein function

Question 3

What is involved in 1. Predicting and MARKING the position of genes and OTHER elements on a genome sequence (in particular with RNA features and protein coding genes) ?

Answer 3

• RNA features: Predict function and location SIMULTANEOUSLY
• Protein coding genes: Predict the location of genes on genome (Gene Finders)
• Translate the encoded protein and predict function

Question 4

What is involved in 2. Predicting protein function?

Answer 4

• Similarity to the characterised proteins

- “Hypothetical Proteins” (not similar to any characterized protein)

Question 5

What are features of protein coding genes in prokaryotes?

Answer 5

• Contained in an ORF (Sequence b/w INITIATION and STOP codons, >50bp are ideal)
• Initiation codon, Ribosome binding site, Minimum length

Question 6

Can prokaryotes contain introns?

Answer 6

-Yes but they are rare and can be polyscistronic (one gene: many proteins)

Question 7

What is usually the initiation codon in prokaryotes?

Answer 7

• ATG (90%) –> Met
• GTG (8%) –> Val
• TTG (1%) –> Leu

Question 8

Why aren’t ALL ORFS marked as genes?

Answer 8

• Because if there are multiple potential ORFs with lots of stop codons (top) BUT there is a clear gene ‘overlap’ where a region ACTUALLY CODES for a gene, that will be the coding one
• Gene overlap is VERY RARE so generally only the largest one will be coding ORF marked as a gene

Question 9

Is gene overlap for coding genes common?

Answer 9

• NO it is RARE

Question 10

What are Gene Finders?

Answer 10

• Programs such as GeneMarkS, GLIMMER, Prodigal (find all ORFs in gene)

Question 11

What do Gene Finders do?

Answer 11

• Identify a potential START codon (ATG but also GTG and TTG)
• They check for CONTEXT with the RIBOSOME BINDING SITE

Question 12

How can we predict protein function? (2 tools)

Answer 12

1. Databases–> For protein sequences and function (GenBank)
2. Using sequence SIMILARITY to predict function–> Proteins with almost same sequence are likely to have the same/similar function

Question 13

What is BLASTp used for?

Answer 13

• Protein query versus protein database

Question 14

What is BLASTx used for?

Answer 14

• Nucleotide query versus protein database (translated into 6 peptide sequences for each of the 6 reading frames)

Question 15

What does the ‘Expect’ section in BLAST mean?

Answer 15

-That the likelihood of the match happening by chance is very close to zero (0.1 cut off generally)

Question 16

What do the identities and positives mean?

Answer 16

-23% identical residues and 43% similar residues respectively

Question 17

What does it mean if there is a less than 10% identity between query sequence and database?

Answer 17

• Similarity occurs by CHANCE (NOT RELATED)

Question 18

What does it mean if there is a 10-35% identity

Answer 18

• MIGHT have related function but still low..

Question 19

What does it mean if there is a less than >35% identity between query sequence and database?

Answer 19

• Probably have a related function

Question 20

Can the difference of just ONE amino acid change, cause a strain of P.multocida to be virulent?

Answer 20

• YES!
• Change from Leucine to Serine
• changes Fis gene–> makes protein capsule –> contributes to virulence
• Can prove it by complementation (putting the virulent strain into the avirulent strain and observing restoration of phenotype (capsule)