MOLECULAR INFORMATION FLOW AND PROTEIN PROCESSING

CHAPTER 6

gene and genome

gene - smallest unit that can be expressed

genome - all genetic elements

what is in 2’ of DNA and RNA

DNA - hydrogen atom - H group

RNA - OH - hydroxyl group - OH group

nucleotide vs nucleoside

• tide - contains phosphate group

  • side - does not contain phosphate group

Pyrimidines vs Purines

Pyrimidines - thymine, uridine, cytosine

Purines - adenine and guanine

structure of DNA

Backbone - alternating phosphate group and pentose sugar deoxyribose

• phosphodiester bonds connect them together

Hydrogen bonds - link nucleotides together

• Primary structure - nucleotides that encode genetic information

• antiparallel

• major groove - allows proteins to read and interact with genetic code

• minor groove - binding site for structural proteins

  • semiconservative

types of RNA

mRNA (messenger RNA) - carries information to ribosome

tRNA (transfer RNA) - converts mRNA information to amino acid sequences

rRNA - structure ribosome components

Basic steps of information flow

Replication - DNA is duplicated by DNA polymerase

Transcription - information from DNA is transferred to RNA by RNA polymerase

translation - mRNA builds polypeptides on ribosomes

RNA polymerase

binds to promoter unwinding double helix

• reads 3-5 prime but replicates 5’-3’

Primer and primase

primer - short fragment that provides essential free 3’-OH group required for DNA polymerase

primase - RNA polymerase enzyme that makes primers on single stranded DNA to start replication

deoxyribonucleotide triphosphate (dNTPs)

DNA monomers/ DNA building blocks of nucleotides of DNA

DNA polymerase 3 and 1

DNA pol 3 - primary replicating enzyme replicating chromosomal DNA

DNA pol 1 - lesser replication role focused on cleanup and repair

important parts of bacterial DNA replication

ORIc - origin of replication in prokaryotes, fixed sequence of nucleotides on DNA strand that start replication

• only one origin

Replication fork - Y - shaped structure where DNA is unzipped becoming single stranded

• DNA Helicase - enzyme that unwinds double helix

  • adds single strand binding proteins to keep DNA strands open

Leading and lagging strand

leading - new continuous DNA made towards the replication fork

Lagging strand - new discontinuous DNA made moving away from the replication fork

• short fragments known as Okazaki fragments

  • DNA ligase - stitches Okazaki fragments together

Steps of DNA replication of lagging strand

1. Helicase unwinds double helix

2. Priming - primase adds a short RNA primer to make staring point for DNA polymerase

3. Elongation - DNA poly 3 builds the Okazaki fragments

• add DNA bases from 3’ end of primer

4. excision - DNA poly 1 removes temporary RNA primer from strand using 5’-3’ exonuclease

5. filling - DNA polymerase fills in empty gap with correct DNA base sequence

   1. Ligase - DNA ligase glues Okazaki fragments together making 1 continuous strand

Theta structure of circular DNA

theta like shape on bacterial chromosome forms bi-directional replication fork

Replisome

protein factory linking helicase, primase, and polymerase together to synthesize DNA strands simultaneously at replication fork

what enzyme activity is responsible for proofreading

proofreading - limits mistakes via 3’-5’ exonuclease activity in bacterial DNA replication

Transcription

process of using DNA as template to make RNA

Promoters and Strong promoters (2)

DNA sequences where transcription starts

• Pribnow box/TATAAT box - located (-10) 10 bases upstream of transcription start site

  • TTGACA (-35) - base sequence located -35 sequence

what marks the end of transcription

transcription terminators

RNA polymerase and sigma factor

Core enzyme responsible for synthesizing single stranded DNA

• Sigma factor - protein that helps recognize promoter sequence that begins transcription

  • subunit of RNA polymerase

Transcription unit

segment of DNA that is transcribed into 1 RNA molecule

• initiation site, set of instructions and termination site

Opening reading frame

section of mRNA that can be translated to proteins

• start codon, series of instruction, and stop codon

Co-transcribed gene

2 or more genes transcribed together into same molecule

• multiple genes copied together

start and stop codon

start codon - AUG

stop codon - UGA UAG UAA

Operons

group of bacterial genes that are controlled together and transcribed into one RNA molecule

• group of genes that work together and are turned on or off together

Polycistronic mRNA

1 messenger RNA molecule that contains multiple Open reading frames and can encode multiple proteins

• POLY - many

• CISTRONIC - coding regions

  • one mRNA contains instructions for several proteins

    • ORFs - sequence of mRNA that can be translated into protein

Transcription in Eukarya

process of using DNA as template to make mRNA

• carried out by RNA polymerase reading template strand making complementary 5’-3’ RNA strand

• takes place in nucleus

TATA box

specific DNA sequence found in promoter region of eukaryotic genes

• 6-8 base pairs long

• 18-27 nucleotides upstream from start of transcription

• recognized by TATA-binding protein - helps RNA polymerase begin transcription

RNA splicing

eukaryotic genes contain both coding and noncoding regions

• exons - coding sequences kept in mature RNA

• introns - noncoding sequences that are removed during RNA processing

  • RNA splicing - process of removing introns and joining exons together - mandatory before undergoing translation

Primary transcript

original, unprocessed RNA

• contains introns and exons

• before RNA splicing occurs

Spliceosome

made of RNA and proteins and carries out RNA splicing in the eukaryotic nucleus

• removes introns from primary transcript, and joins exons together to make mature RNA

RNA capping

addition of a methylated guanine to the 5’ end of mRNA

• occurs before splicing and is needed to initiate translation

Polyadenylation/Poly(A) tail

addition of adenine to the 3’ end of mRNA

• contains approximately 100-200 adenylate resiudes to stabilize mRNA

  • must be removed before mRNA can be degrade

job of 5’ cap and 3’ poly A tail

protect mRNA from degradation, protects mRNA when transferring from nucleus to ribosome

Overall process

1. DNA is transcribed by RNA polymerase

2. Primary transcript containing exons and introns

3. RNA processing and addition of 5’ cap

4. splicing by spliceosome removal of introns and exons join

5. enzyme cuts primary transcript and immediately attach poly(A) tail

6. mature mRNA ready for translation

Protein structures

Primary structure - linear sequence of amino acids

Secondary structures - formed by hydrogen bonding (alpha-helix or beta sheets)

Tertiary structure - 3-D shape due to folding of single polypeptide chain

Quaternary structure - assembly of multiple folded polypeptide subunits

Genetic code

set o rules that determines which amino acid is specified by a sequence of 3 RNA bases

• 3 bases (codon) 64 possible codons

  • instructions to translate mRNA codons into amino acids

Degenerate code

more than one codon can code for the same amino acid

Anticodon

sequence of 3 bases found on tRNA pairing with complementary codon on mRNA

• allows tRNA to bring the correct amino acid to the ribosome during translation

Wobble

irregular type of base pairing allowed at the third position of the codon

• allows some tRNAs to recognize more than one codon

  • explains why multiple codons make the same amino acid by relaxing the third position of the codon

Reading Frame

how mRNA is divide into groups of 3 bases to be read as a codon

• must begin at the correct nucleotide, if reading frame shifts, codons change and can produce different amino acid sequence

Shine-Dalgarno sequence/ ribosome-binding site

• ensures ribosome starts translation at proper reading frames in bacteria, not eukaryotes

Ribosomes

large complexes of proteins and RNA where proteins are biosynthesized

Steps of Protein synthesis

1. initiation - ribosome assembles on the mRNA at the AUG start codon with the initiator tRNA

2. Elongation - tRNAs bring amino acids to the ribosome, which joins them together to grow the polypeptide chain

3. Termination - ribosome reaches a stop codon, release factor releases the completed polypeptide, and the ribosomes separates

sites on the ribosome (3)

A site (accept) - incoming tRNA carrying a new amino acid enters

P site (Peptide) - holds the tRNA carrying the growing polypeptide chain

E site (Exit) - Empty tRNA exits the ribosome

Release factor

recognizes stop codon and cleave polypeptide from tRNA

Polysomes

a complex formed by multiple ribosomes simultaneously translating a single mRNA

how many RNA polymerases do eukaryotes have

Eukaryotes have multiple RNA polymerases that make different types of RNA

Does eukaryotic mRNA have a ribosome-binding site

No because eukaryotes make ribosomes enter right into the mRNA

how is eukaryotic mRNA recognized by the ribosome

Eukaryotic mRNA is recognized by its modified 5’ guanine cap, which helps initiate translation